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Abstract
As small non-coding regulatory RNAs, microRNAs are capable of silencing gene expression by translational repression or mRNA degradation. Accumulating evidence indicates that deregulation of microRNAs is often associated with human malignancies and suggests a causal role of microRNAs in neoplasia, presumably because microRNAs can function as oncogenes or tumor suppressors. Among them, miR-205 is significantly underexpressed in breast tumors compared with matched normal breast tissue although miR-205 has been shown to be upregulated in some other type of tumors. Furthermore, breast cancer cell lines, including MCF-7 and MDA-MB-231, express a lower level of miR-205 than the non-malignant MCF-10A cells. Ectopic expression of miR-205 significantly inhibits cell proliferation and anchorage-independent growth as well as cell invasion. These findings establish the tumor suppressive role of miR-205, which is probably through direct targeting of oncogenes such as ErbB3 and Zeb1. Therefore, miR-205 may serve as a unique therapeutic target for breast cancer.
... Various tumor-associated specific molecular markers, such as miRs, are important in the early diagnosis of cancer and other diseases (5). miR expression in cancer tissues is dysregulated (2,3,6), and their expression pattern exhibits a certain degree of tissue specificity (7,8). miR-205 is a multi-functional gene located in the 1q32.2 ...
... miR-205 is a multi-functional gene located in the 1q32.2 locus of the human genome, and is involved in various physiological and pathological processes, including tumorigenesis, inflammation and immunity (8). miR-205 is abnormally expressed in a variety of malignant tumors and its expression is closely related to the incidence and development of tumors, such as head and neck cancer, ovarian cancer and breast cancer (8)(9)(10). ...
... locus of the human genome, and is involved in various physiological and pathological processes, including tumorigenesis, inflammation and immunity (8). miR-205 is abnormally expressed in a variety of malignant tumors and its expression is closely related to the incidence and development of tumors, such as head and neck cancer, ovarian cancer and breast cancer (8)(9)(10). Previously, Lebanony et al (11) and the authors (12) both reported that miR-205 expression is significantly increased in non-small cell lung cancer (NSCLC) tissues, and that it is associated with tumor differentiation grade (12). ...
Previous studies have demonstrated that microRNA (miR)-205-5p expression is significantly increased in non?small cell lung cancer tissues and is associated with tumor differentiation grade. The aim of the present study was to explore the effects of miR?205?5p on viability, apoptosis and invasion of lung cancer A549 cells. The hsa?miR?205?5p small interfering RNA (siRNA) inhibitor was transfected into A549 cells and expression of miR?205?5p was detected by reverse transcription?quantitative polymerase chain reaction (RT?qPCR). Cell viability, apoptosis and invasion were assayed by Cell Counting kit?8, Annexin V/propidium iodide double staining and Transwell assay, respectively. Target genes of miR?205?5p were predicted using bioinformatics analysis. Expression of mRNA and protein levels of candidate target genes following miR?205?5p inhibition were detected using RT?qPCR and western blot analysis respectively. The results demonstrated that relative survival rates of A549 cells were significantly inhibited in miR?205?5p siRNA?transfected cells at 24 and 48?h compared with control cells. Apoptosis was markedly increased in the miR?205?5p siRNA cells compared with control cells. The number of invaded cells following miR?205?5p siRNA silencing was significantly decreased compared with control cells. Bioinformatics analysis revealed that erb?B2 receptor kinase 3 (erbB3), zinc finger E?box binding homeobox 2 (ZEB2), clathrin heavy chain (CLTC) and mediator complex subunit 1 (MED1) may be potential target genes of miR?205?5p. Reduced expression of miR?205?5p significantly increased the expression of ZEB2 mRNA and protein, inhibited the expression of erbB3 protein, but had no significant effect on the expression levels of CLTC and MED1. In summary, reduced expression of miR?205?5p promoted apoptosis and inhibited proliferation and invasion in lung cancer A549 cells through upregulation of ZEB2 and downregulation of erbB3. The present results suggested that the increased miR?205?5p expression observed in non?small cell lung cancer tissues may contribute to increased proliferation and invasion of lung cancer cells and thus to cancer progression.
... Genetic, morphological and IHC features of BLBC were reported, however, there was no universal definition and specific biomarker which could identify those tumors in routine diagnostics (41). Previously studies revealed miR dysregulation and dysfunction in breast cancer (10)(11)(12), and miR-205 was significantly under-expressed in breast tumors compared with matched normal breast tissue (42). In breast cancer cell lines, including MCF-7 and MDA-MB-231, miR-205 expressed lower levels than non-malignant MCF-10A cells (42). ...
... Previously studies revealed miR dysregulation and dysfunction in breast cancer (10)(11)(12), and miR-205 was significantly under-expressed in breast tumors compared with matched normal breast tissue (42). In breast cancer cell lines, including MCF-7 and MDA-MB-231, miR-205 expressed lower levels than non-malignant MCF-10A cells (42). Only a few studies which associated with triplenegative breast cancer revealed that miR-205 was directly transactivated by oncosuppressor p53 (43). ...
... Each miR is capable of regulating hundreds of proteincoding genes. Previous studies revealed that most miR-205 target genes were Her-2/3 and ZEB1/2/3 in breast cancer (42,44). Some others studies also identified that miR-205 directly targeted phosphatase and tensin homolog deleted on chromosome ten and interleukin-24 in A549 cells and human KB oral cancer cells (45). ...
We investigated microRNAs (miRs) specific to its target gene and exerting distinct biological functions for basal-like breast carcinoma (BLBC). Total RNA was extracted and subjected to miR microarray and bioinformatics analysis. Based on the comprehensive analysis, expression of miRs including its target was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot analysis and immunohistochemistry (IHC). Further functional analyses were conducted including proliferation, invasion and apoptosis. miR-205 was identified as downregulated (less than 0.5-fold) in BLBC relatively to normal control (NC). Gene ontology (GO) analysis suggested miR-205 may directly targeted Krüppel-like factor 12 (KLF12; degree=4). Luciferase assay revealed miR-205 directly targeted KLF12 through binding its 3'-untranslated region (3'-UTR; p=0.0016). qRT-PCR and western blot analysis showed miR-205 expression was low in cells (p=0.007) and tumor tissues (n=6; p=0.0074), and KLF12 RNA/protein was observed at high levels in cells (p=0.0026; p=0.0079) and tumor tissues (n=9; p=0.0083); knock-up of miR-205 increased its expression (p=0.0021) but reduced KLF12 RNA/protein levels (p=0.0038; p=0.009) in cells. Modulation of miR-205 expression by transfecting its mimics in cells, was involved in invasion (p=0.00175) and apoptosis (p=0.006). In conclusion, our results supported that miR-205 was a miR specific to BLBC which functioned as tumor suppressor gene through directly targeting and negatively regulating proto-oncogene KLF12. miR-205 dysregulation was involved in invasion and apoptosis. miR-205 and KLF12 provided a potential diagnosis biomarker and therapeutic approach for BLBC.
... One of the upregulated genes found in the list was MIR205HG. This microRNA has been described as a tumour suppressor gene in different tumour types (Song and Bu, 2009; Wu et al, 2009; Majid et al, 2011), a reason whereby we decided to further study its role in lung cancer. miR-205 expression in H358 and H441 shCtrl and shTMP4 cell clones was validated by qPCR (Supplementary Figure 1B). ...
... As expected, the majority of genes with a differential expression were related with cell invasion and motility, in keeping with the functional role described for this serine protease in some tumour types (Jung et al, 2008; Kim et al, 2010). One of the upregulated genes was miR-205, a microRNA that has been described as a tumour suppressor (Song and Bu, 2009; Wu et al, 2009; Majid et al, 2011). ...
... These miRs act by directly targeting the 3 0 -UTR sequence of ZEB1 and SIP1 (Gregory et al, 2008; Park et al, 2008). miR-205 has also been shown to suppress metastatic spread of human breast cancer xenografts in nude mice (Iorio et al, 2009) and to exert a tumour suppressor role by targeting HER3 receptor and VEGF-A in breast cancer (Iorio et al, 2009; Wu and Mo, 2009) and Src in renal cancer (Majid et al, 2011). In lung cancer, overexpression of miR-205 in cell lines hinders cell migration and invasion (Song and Bu, 2009) and its inhibition results in the acquisition of cancer stem cell and EMT properties, which favors tumour progression (Tellez et al, 2011). ...
Background:
TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown.
Methods:
miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC.
Results:
miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels.
Conclusion:
We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.
... Recently, HER2 and HER3 (erbB2 and erbB3), which significantly correlate with breast cancer and poor prognosis, have been shown to be suppressed by miR-125a, miR-125b and miR-205 in breast cancer cells, leading to a reduction in cell proliferation and migration and increased apoptosis (30). Moreover, the suppressive role of miR-205 is mediated through the direct targeting of oncogenes such as Zeb1 (31). It has been shown that the overexpression of miR-145 inhibits estrogen receptor-α (ER-α) protein expression (32) and reduces RTKN protein expression (33), thus reducing breast cancer cell growth and inducing apoptosis, indicating its tumor-suppressive functions. ...
... miR-31 has also been shown to trigger metastatic regression in the lungs by eliciting cell cycle arrest and apoptosis, which can be explained by the miR-31-mediated suppression of integrin-α5, radixin, RhoA (41), the actin cytoskeleton remodeling protein WAVE3 (42) and protein kinase C epsilon (PKC-ε) (43). Other cancer-suppressing miRNAs such as miR-125a, miR-125b and miR-205 significantly inhibit breast cancer cell invasion by directly targeting HER2 and HER3 (30), whereas miR-205 can target Zeb1 (31). Additionally, miR-145 significantly suppresses cell invasion and lung metastasis by directly targeting the metastasis gene mucin 1 (MUC1), leading to a reduction of β-catenin and adherin 11 (44). ...
MicroRNAs (miRNAs) are non-coding single-stranded RNAs in eukaryotes and are involved in the regulation of the post-transcriptional expression of specific genes. Studies have demonstrated that miRNAs play important roles in regul-ating diverse physiological events such as cell proliferation, differentiation and embryo development. In recent decades, considerable attention has been given to the relationship between miRNA and the pathology of cancers, particularly breast cancer. A large number of miRNAs have been shown to be involved in the pathophysiology of breast cancer. Studies have revealed that some miRNAs might regulate the oncogenesis and growth of breast cancer by acting on breast tumor-initiating cells or other downstream targets. Studies have also demonstrated that some miRNAs act as suppressors of metastasis or promoters of breast cancer. Additionally, certain miRNAs are involved in cancer tissue angiogenesis (one of the most important mechanisms of tumor growth and metastasis). Clinical evidence indicates that some miRNAs can be used as diagnostic and prognostic biomarkers for breast cancer due to their significantly increased or decreased expression in cancer tissue. Moreover, certain miRNAs may have therapeutic potential for targeting ER-α/HER, breast tumor-initiating cells and metastasis as well as multidrug resistance. In this review, we discuss the relationship between miRNAs and the pathogenesis of breast cancer as well as the progress of current research on the miRNA-specific diagnosis, prognosis and treatment of breast cancer.
... miR-205 is found exclusively in normal ducts and lobular myoepithelial cells of the breast but is significantly reduced in breast tumor tissues [15,16]. The purported tumor suppressive functions of miR-205 in breast cancer is due to direct targeting of several oncogenes such as VEGFA, E2F1, E2F5, PKC epsilon and HER3 reviewed in 16 as well as attenuating epithelial to mesenchymal transition (EMT) by suppressing ZEB1 and ZEB2161718. The intent of this study was to identify additional target genes for miR-205 that may be involved in the aggressive phenotype of TNBC. ...
... A link between HMGB3 and the invasive, metastatic phenotype has been made by demonstrating that HMGB3 mRNA levels are increased in metastatic breast cancers (Figure 5A), that knockdown of HMGB3 decreases in vitro invasiveness (Figure 4B) and patients with increased HMGB3 expression have poor survival (Figure 5E). Moreover, we link the up-regulation of HMGB3 in breast cancer to miR-205, a miRNA that has been shown here (Figure 1) and in other studies14151618] to have reduced expression in breast cancer. The HMG superfamily of DNA binding proteins recognize the HMG box, typically located in promoters. ...
Identifying targets of dysregulated microRNAs (miRNAs) will enhance our understanding of how altered miRNA expression contributes to the malignant phenotype of breast cancer. The expression of miR-205 was reduced in four breast cancer cell lines compared to the normal-like epithelial cell line MCF10A and in tumor and metastatic tissues compared to adjacent benign breast tissue. Two predicted binding sites for miR-205 were identified in the 3' untranslated region of the high mobility group box 3 gene, HMGB3. Both dual-luciferase reporter assay and Western blotting confirmed that miR-205 binds to and regulates HMGB3. To further explore miR-205 targeting of HMGB3, WST-1 proliferation and in vitro invasion assays were performed in MDA-MB-231 and BT549 cells transiently transfected with precursor miR-205 oligonucleotide or HMGB3 small interfering RNA (siRNA). Both treatments reduced the proliferation and invasion of the cancer cells. The mRNA and protein levels of HMGB3 were higher in the tumor compared to adjacent benign specimens and there was an indirect correlation between the expression of HMGB3 mRNA and patient survival. Treatment of breast cancer cells with 5-Aza/TSA derepressed miR-205 and reduced HMGB3 mRNA while knockdown of the transcriptional repressor NRSF/REST, reduced miR-205 and increased HMGB3. In conclusion, regulation of HMGB3 by miR-205 reduced both proliferation and invasion of breast cancer cells. Our findings suggest that modulating miR-205 and/or targeting HMGB3 are potential therapies for advanced breast cancer.
... After a series of bioinformatics analyses and external experimental verification, upregulated miR-205-5p and downregulated PTPRM in tumor tissues were finally screened out. The expression of miR-205-5p is closely associated with the incidence and development of tumors, such as head and neck cancer, ovarian cancer and breast cancer (Iorio et al., 2007;Tran et al., 2007;Wu and Mo 2009). It has been reported that the expression of miR-205 in non-small cell lung cancer (NSCLC) tissues is significantly increased and is related to the degree of tumor differentiation, which may lead to increased proliferation and invasion of lung cancer cells, thus leading to cancer progression (Lebanony et al., 2009;Jiang et al., 2013;Duan et al., 2017;Jiang et al., 2017). ...
Purpose: MicroRNA (miRNA) binds to target mRNA and inhibit post-transcriptional gene expression. It plays an essential role in regulating gene expression, cell cycle, and biological development. This study aims to identify potential miRNA-mRNA regulatory networks that contribute to the pathogenesis of lung squamous cell carcinoma (LUSC).
Patients and Methods: MiRNA microarray and RNA-Seq datasets were obtained from the gene expression omnibus (GEO) databases, the cancer genome atlas (TCGA), miRcancer, and dbDEMC. The GEO2R tool, “limma” and “DEseq” R packages were used to perform differential expression analysis. Gene enrichment analysis was conducted using the DAVID, DIANA, and Hiplot tools. The miRNA-mRNA regulatory networks were screened from the experimentally validated miRNA-target interactions databases (miRTarBase and TarBase). External validation was carried out in 30 pairs of LUSC tissues by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Receiver operating characteristic curve (ROC) and decision curve analysis (DCA) were conducted to evaluate the diagnostic value. Clinical, survival and phenotypic analysis of miRNA-mRNA regulatory networks were further explored.
Results: We screened 5 miRNA and 10 mRNA expression datasets from GEO and identified 7 DE-miRNAs and 270 DE-mRNAs. After databases screening and correlation analysis, four pairs of miRNA-mRNA regulatory networks were screened out. The miRNA-mRNA network of miR-205-5p (up) and PTPRM (down) was validated in 30 pairs of LUSC tissues. MiR-205-5p and PTPRM have good diagnostic efficacy and are expressed differently in different clinical features and are related to tumor immunity.
Conclusion: The research identified a potential miRNA-mRNA regulatory network, providing a new way to explore the genesis and development of LUSC.
... The first disease in which a link with a microRNAs dysfunction was established was chronic lymphocytic leukemia [12]. Recent studies have shown the role of miR-205 in inhibiting the development of metastases in breast cancer [13]. In classical Hodgkin lymphoma, miR-21, miR-494, and miR-1973 present in blood plasma serve as markers of the disease [14]. ...
This study aimed to investigate the expression level of miR-126 in children with psoriasis in the epidermis affected by psoriasis and intact buccal epithelium, establish the impact on the characteristics of the course of psoriasis and the results of therapy in children with psoriasis of initial expression levels of miR-126. miR-126 expression levels in psoriatic keratinocytes and buccal epithelium were determined in 54 children with psoriasis on the severity of psoriasis, treatment efficacy. miR-126 levels in the buccal epithelium in children with psoriasis were reduced compared to healthy children (AUC=0.776±0.048, p<0.001). There were no discrepancies between miR-126 expression levels in psoriatic keratinocytes and buccal epithelium (p=0.097). There are statistically significant discrepancies between miR-126 expression levels in the psoriatic epidermis depending on the clinical form of psoriasis (AUC=0.637±0.056; p=0.014) and severity according to BSA (AUC=0.634±0.063; p=0.034). Depending on the miR-126 level in the buccal epithelium, the response to treatment (PASI<75) in children with high miR-126 is worse than in children with expected miR-126 levels (OR 2.79; 95%; CI: 1.19 - 6.51). Treatment failures were observed in children with high levels of miR-126 in the buccal epithelium compared to miR-126 in the psoriatic epidermis: children aged 12/13 to 17 years (OR 2.44; 95% CI: 1.02 - 5.85), children with PGA=4 (OR 3.16; 95% CI: 1.34 - 7.43). The location and level of miR-126 expression affects the course of psoriasis and the outcome of treatment. High levels of miR-126 in psoriatic keratinocytes lead to manifestations of plaque psoriasis with a course of moderate to severe forms. Initial miR-126 levels in the buccal epithelium in children with psoriasis are a prognostic criterion for response to therapy and can be used as a marker for prescribing systemic treatment.
... The detection of microRNA is relevant due to recent studies considering the use of microRNAs as promising biomolecular markers associated with the development of various pathologies in humans [21][22][23]. In this way, the use of miR1246, miR-4634, miR1307-3p, miR-6875-5p, miR-6861-5p, and miR-10b as potential markers of breast cancer has been discussed [23,24]. With regard to ASDs, in the literature, a number of microRNAs, including miR-106a, miR-494, miR-19a, miR-19b, etc. [25], were reported to be associated with these disorders. ...
Gas-phase etching and optical lithography were employed for the fabrication of a silicon nanoribbon chip (Si-NR chip). The quality of the so-fabricated silicon nanoribbons (Si-NRs) was monitored by optical Raman scattering spectroscopy. It was demonstrated that the structures of the Si-NRs were virtually defect-free, meaning they could be used for highly sensitive detection of biological macromolecules. The Si-NR chips were then used for the highly sensitive nanoelectronics detection of DNA oligonucleotides (oDNAs), which represent synthetic analogs of 106a-5p microRNA (miR-106a-5p), associated with the development of autism spectrum disorders in children. The specificity of the analysis was attained by the sensitization of the Si-NR chip sur-face by covalent immobilization of oDNA probes, whose nucleotide sequence was complementary to the known sequence of miR-106a-5p. The use of the Si-NR chip was demonstrated to al-low for the rapid label-free real-time detection of oDNA at ultra-low (~10−17 M) concentrations.
... Moreover, the up-regulated miR-205 can also target ZEB1 to inhibit breast cancer cell invasion [32]. However, restoration of miR-205 expression in breast cancer can act on Eerb B3 and VEGF-A genes to inhibit the proliferation and invasion of breast cancer cells [33]. This results was consistent to our work. ...
MicroRNA-205 (miR-205) is believed to be related to the progress of tumors. HOXD9 has been proved to be expressed abnormally in several kinds of cancers. However, the role of miR-205 and HOXD9 in breast cancer remains unclear. The biological role of miR-205 in breast cancer cell proliferation and chemoresistance was investigated. The expression of miR-205 in clinical tissues and breast cancer cell lines were analyzed using quantitative real-time PCR test (qRT-PCR). Overexpression and knockdown models of miR-205 were established to study cell proliferation and chemotherapy-resistant. Moreover, the potential relationships between miR-205 and HOXD9/Snail1 were measured using qRT-PCR, western blot, and chemotherapy-resistant study. miR-205 was lowly expressed in breast cancer tissues and cell lines. Overexpression of miR-205 could inhibit cell proliferation and chemotherapy-resistance. Moreover, we proved that miR-205 could target the HOXD9-Snail1 axis to suppress triple negative breast cancer cell proliferation and chemoresistance. The activation of Snail1 gene by HOXD9 was also proved in this study. The present study may provide a novel insight for the therapeutic strategies of breast cancer through targeting miR-205/HOXD9/Snail1.
... This tumor suppressor miRNA, known to be exclusively expressed in myoepithelial cells, has been associated with the regulation of adherens junctions and focal adhesion. As such, downregulation of hsa-miRNA-205-5p leads to tumor invasion [46][47][48]. Two shared miRNAs between NAF and milk were hsa-miRNA-200b-3p and hsa-miRNA-200c-3p, belonging to the tumor suppressor miRNA-200 family [49,50]. ...
Background:
MicroRNAs (miRNAs) target 60% of human messenger RNAs and can be detected in tissues and biofluids without loss of stability during sample processing, making them highly appraised upcoming biomarkers for evaluation of disease. However, reporting of the abundantly expressed miRNAs in healthy samples is often surpassed. Here, we characterized for the first time the physiological miRNA landscape in a biofluid of the healthy breast: nipple aspirate fluid (NAF), and compared NAF miRNA expression patterns with publically available miRNA expression profiles of healthy breast tissue, breast milk, plasma and serum.
Methods:
MiRNA RT-qPCR profiling of NAF (n = 41) and serum (n = 23) samples from two healthy female cohorts was performed using the TaqMan OpenArray Human Advanced MicroRNA 754-Panel. MiRNA quantification data based on non-targeted or multi-targeted profiling techniques for breast tissue, breast milk, plasma and serum were retrieved from the literature by means of a systematic search. MiRNAs from each individual study were orderly ranked between 1 and 50, combined into an overall ranking per sample type and compared.
Results:
NAF expressed 11 unique miRNAs and shared 21/50 miRNAs with breast tissue. Seven miRNAs were shared between the five sample types. Overlap between sample types varied between 42% and 62%. Highly ranked NAF miRNAs have established roles in breast carcinogenesis.
Conclusion:
This is the first study to characterize and compare the unique physiological NAF-derived miRNA landscape with the physiological expression pattern in breast tissue, breast milk, plasma and serum. Breast-specific sources did not mutually overlap more than with systemic sources. Given their established role in carcinogenesis, NAF miRNA assessment could be a valuable tool in breast tumor diagnostics.
... Moreover, the miR-200 family members and miR-205 inhibit epithelial to mesenchymal transition (EMT) via E-cadherin regulation and ZEB1 inhibition [23]. The known tumor suppressive functions of miR-205 in breast cancer include direct repression of several oncogenes such as VEGFA and HER3, as well as suppression of EMT by inhibiting ZEB1 and ZEB2 expression [36,38,39]. ...
Previous studies have shown that transglutaminase 2 (TG2) induces epithelial to mesenchymal transition (EMT) in various tumors. Several studies have also demonstrated the critical role of microRNAs (miRNAs) in regulating EMT of various types of tumors. However, the relationship between TG2 and miRNAs is not well understood. In the present study, we investigated if miR-205, which is known to inhibit EMT and is commonly regulated by TG2, contributes to TG2-induced EMT of human breast cancer cells. We have analyzed the expression of miR-205 in TG2-expressing and TG2-non-expressing breast cancer cells by quantitative real-time PCR (qRT-PCR) and the expression of TG2 and EMT related markers, such as ZEB1 and Vimentin, by western blotting. We also have studied the regulation of tumor metastasis by miR-205 and TG2 using matrigel invasion assays, intracardiac injection of breast cancer cells into mice and in vivo bioluminescent imaging. MiR-205 was significantly downregulated in high TG2-expressing or TG2-transfected breast cancer cells than in low TG2-expressing or mock-transfected breast cancer cells. Overexpression of miR-205 reduced the bone metastasis of MCF7/TG2-C277S cells that express transamidase-activity deficient TG2 and inhibits the invasiveness of MDA-MB-231 breast cancer cells that express TG2. Bioluminescent imaging showed that intracardiac injection of MCF7/TG2-C277S cells in mice promoted bone tumors, especially in the knee and jaw, but MCF7/TG2-C277S cells ectopically expressing miR-205 did not metastasize. The GTP binding activity, but not transamidase activity, of TG2, induces EMT in breast cancer cells by inhibiting the expression of miR-205 that suppresses EMT by downregulating the expression of ZEB1, an EMT marker. Moreover, in vivo experiments demonstrate that miR-205 down-regulation by TG2 induces bone metastasis of breast cancer cells.
... Besides, the miRNA-breast neoplasms associations have been verified by many previous works of literature. For example, miR-21 has been found to be excessive in breast neoplasms [38], while miR-429 and miR-200c are down-regulated [39]. Similarly, we sorted the final prediction results according to the prediction score. ...
Background:
As an important non-coding RNA, microRNA (miRNA) plays a significant role in a series of life processes and is closely associated with a variety of Human diseases. Hence, identification of potential miRNA-disease associations can make great contributions to the research and treatment of Human diseases. However, to our knowledge, many existing computational methods only utilize the single type of known association information between miRNAs and diseases to predict their potential associations, without focusing on their interactions or associations with other types of molecules.
Results:
In this paper, we propose a network embedding-based method for predicting miRNA-disease associations by preserving behavior and attribute information. Firstly, a heterogeneous network is constructed by integrating known associations among miRNA, protein and disease, and the network representation method Learning Graph Representations with Global Structural Information (GraRep) is implemented to learn the behavior information of miRNAs and diseases in the network. Then, the behavior information of miRNAs and diseases is combined with the attribute information of them to represent miRNA-disease association pairs. Finally, the prediction model is established based on the Random Forest algorithm. Under the five-fold cross validation, the proposed NEMPD model obtained average 85.41% prediction accuracy with 80.96% sensitivity at the AUC of 91.58%. Furthermore, the performance of NEMPD is also validated by the case studies. Among the top 50 predicted disease-related miRNAs, 48 (breast neoplasms), 47 (colon neoplasms), 47 (lung neoplasms) were confirmed by two other databases.
Conclusions:
The proposed NEMPD model has a good performance in predicting the potential associations between miRNAs and diseases, and has great potency in the field of miRNA-disease association prediction in the future.
... In addition to its role in regulating Kir4.1, miR-205 has been implicated as an oncogene or tumor suppressor depending on the target they regulate [17]. When miR-205 was overexpressed in prostate cancer cells, these cells showed a reduction in invasion and cell locomotion [18]. ...
Protecting neurons from neurotoxicity is a job mainly performed by astrocytes through glutamate uptake and potassium buffering. These functions are aided principally by the Kir4.1 inwardly rectifying potassium channels located in the membrane of astrocytes. Astrocytes grown in hyperglycemic conditions have decreased levels of Kir4.1 potassium channels as well as impaired potassium and glutamate uptake. Previous studies performed in a human corneal epithelial cell injury model demonstrated a mechanism of regulation of Kir4.1 expression via the binding of microRNA-250 (miR-205) to the Kir4.1 3´ untranslated region. Our purpose is to test if astrocytes express miR-205 and elucidate its role in regulating Kir4.1 expression in astrocytes grown in hyperglycemic conditions. We used quantitative-PCR to assess the levels of miR-205 in astrocytes grown in high glucose (25 mM) medium compared to astrocytes grown in normal glucose (5 mM). We found that not only was miR-205 expressed in astrocytes grown in normal glucose, but its expression was increased up to six-fold in astrocytes grown in hyperglycemic conditions. Transfection of miR-205 mimic or inhibitor was performed to alter the levels of miR-205 in astrocytes followed by western blot to assess Kir4.1 channel levels in these cells. Astrocytes treated with miR-205 mimic had a 38.6% reduction of Kir4.1 protein levels compared to control (mock-transfected) cells. In contrast, astrocytes transfected with miR-205 inhibitor were significantly upregulated compared to mock by 47.4%. Taken together, our data indicate that miR-205 negatively regulates the expression of Kir4.1 in astrocytes grown in hyperglycemic conditions.
... Gao A. et al., demonstrated the possibility for the specific revelation of lung cancer by NW-biosensor detection of marker miRNA-126 at a concentration of 10 −16 M [12]. The literature contains discussions on the following miRNAs as potential markers: miR1246, miR-4634, miR1307-3p, miR-6875-5p, miR-6861-5p, and (miR)-10b [13,14]. ...
Information about the characteristics of measuring chips according to their storage conditions is of great importance for clinical diagnosis. In our present work, we have studied the capability of chips to detect nanowire biosensors when they are either freshly prepared or have been stored for either one or two years in a clean room. Potential to detect DNA oligonucleotides (oDNAs)—synthetic analogues of microRNAs (miRNAs) 198 and 429 that are associated with the development of prostate cancer (PCa)—in buffer solution was demonstrated using a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). To provide biospecific detection, nanowire surfaces were sensitized with oligonucleotide probes (oDNA probes) complimentary to the known sequences of miRNA 183 and 484. In this study it is demonstrated that freshly prepared SOI-NW biosensor chips with n-type conductance and immobilized oDNA probes exhibit responses to the addition of complimentary oDNAs in buffer, leading to decreases in chips’ conductance at a concentration of 3.3 × 10−16 M. The influence of storage time on the characteristics of SOI-NW biosensor chips is also studied herein. It is shown that a two-year storage of the chips leads to significant changes in their characteristics, resulting in “inverse” sensitivity toward negatively charged oDNA probes (i.e., through an increase in chips’ conductance). It is concluded that the surface layer makes the main contribution to conductance of the biosensor chip. Our results indicate that the detection of target nucleic acid molecules can be carried out with high sensitivity using sensor chips after long-term storage, but that changes in their surface properties, which lead to inversed detection signals, must be taken into account. Examples of the applications of such chips for the detection of cancer-associated microRNAs in plasma samples of patients with diagnosed prostate cancer are given. The results obtained herein are useful for the development of highly sensitive nanowire-based diagnostic systems for the revelation of (prostate) cancer-associated microRNAs in human plasma.
... Reported data revealed that there is a negative correlation between mir-29 and ID1 by c-Myc due to overexpression of c-Myc in cells of lung cancer suppressed the level of mir-29 (19). High level of mir-205 were also reported by various research studies in lung cancer (24), not only in lung cancer but also in other cancer as well (48,49). Additionally, types of lung cancer can be distributed through mir-205 and also regulates the expression of PTEN (50). ...
Lung cancers are one of a leading cause of death in smokers and healthy individuals. The earlier screening and diagnosis of lung cancers is essential for the therapeutic purposes. The miRs can detect in different biological specimens of lung cancer patients. Present review was concentrate on miRs as a diagnostic biomarkers in lung cancer tissues and other body fluid. All the published articles in different international journals wereretrieve from search engines by using terms such as "miRs as biomarker in lung cancer" from 2011-2018. All the relevant articles about lung cancer microRNAs were included. Data analyzed were through Microsoft excel 2016. Several articles were retrieve from various data banks, only twenty seven articles were found relevant to present review in which twenty four were of tissues, two of pleural fluid and one of bronchoalveolar lavage fluids. Hundreds of miRs were dysregulated but 38 were significantly up or down regulated in reported studies. Out of total 38, only six miRs were consistently dysregulated in various studies that are mir-96, mir-183, mir-196a, mir-205, mir-29 and mir-21 in whichmir-96, mir-183, mir-196a, mir-205 and mir-21 were upregulated while mir-29 were downregulted in various studies whereas only single hsa-let-7b-5p were inconsistently reported by two different studies. It is concluded with the result of present review that significantly dysregulated miRs have a crucial role in early diagnosis regarding the therapeutic purpose of lung cancer.
... Ectopic expression of miR-205 significantly inhibits cell proliferation and anchorage-independent growth as well as cell invasion. miR-205 may serve as a unique therapeutic target for BC [29] . At present, we have not developed a drug targeted to a certain miRNA for BC, but our research group is already in progress. ...
Breast cancer (BC) is a malignancy with the highest incidence in women. Great progress has been made in research related to traditional precision medicine for BC. However, many reports have suggested that patients with BC have not benefited a lot from such progress. Thus, we analyze traditional precision medicine strategies for BC, sum up their limitations and challenges, and preliminarily propose future orientations of precision medicine strategies based on a database on drug reaction of patients with BC. According to related research, traditional precision medicine strategies for BC, which are based on molecular subtypes, perform pertinent treatments, new drug research and development according to molecular typing results. Nevertheless, these strategies still have some deficiencies. First, there are very few patients with each molecular subtype, the match ratio of drugs is low. Second, these strategies can not solve the problem of poor drug sensitivity resulting from heterogeneity. The main strategy we put forward in the present paper is based on patients’ varying drug reactions. Focusing on treating existing patients and maximizing the utilization of existing drugs, it is expected to not have deficiencies of traditional precision medicine for BC, including low match rate and poor therapeutic efficacy arising from tumor heterogeneity of BC.
... Some mimics also have longer sequences such as that of miRNA precursors. Recent studies have shown that ectopic expression of miR-205 suppresses tumor growth by efficiently blocking cell proliferation, anchorage-independent growth and cell invasion [336]. Several reports have shown that enhanced expression of miR-16 curbs cell growth and proliferation and promotes apoptosis by downregulating Cyclin D1 and Bcl-2 at mRNA and protein levels in MCF-7 cells [337]. ...
Paradigm shifting studies especially involving non-coding RNAs (ncRNAs) during last few decades have significantly changed the scientific perspectives regarding the complexity of cellular signalling pathways. Several studies have shown that the non-coding RNAs, initially ignored as transcriptional noise or products of erroneous transcription; actually regulate plethora of biological phenomena ranging from developmental processes to various diseases including cancer. Current strategies that are employed for the management of various cancers including that of breast fall short when their undesired side effects like Cancer Stem Cells (CSC) enrichment, low recurrence-free survival and development of drug resistance are taken into consideration. This review aims at exploring the potential role of ncRNAs as therapeutics in breast cancer, by providing a comprehensive understanding of their mechanism of action and function and their crucial contribution in regulating various aspects of breast cancer progression such as cell proliferation, angiogenesis, EMT, CSCs, drug resistance and metastasis. In addition, we also provide information about various strategies that can be employed or are under development to explore them as potential moieties that may be used for therapeutic intervention in breast cancer.
... Consistently, the inhibition of miR-205-PKCε interaction through the use of a miRNA mask almost completely abolished the radiosensitizing effect of the miRNA as a consequence of the complete recovery of PKCε expression. miR-205 reconstitution in experimental models of human breast cancer, another tumor type characterized by a reduced expression of the miRNA [33], was found to improve the radiation response by directly targeting ZEB1 and the ubiquitin-conjugating enzyme Ubc13, thus inhibiting HR-mediated repair of DNA-DSBs [4]. Consistently, in this study we found a significant inhibition of ZEB1 expression, at both mRNA and protein level, following miR-205 reconstitution in PCa cell lines. ...
Background
Radiotherapy is one of the main treatment options for non-metastatic prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a considerable fraction of patients still experience relapse due to the development of resistance. Radioresistance is a complex and still poorly understood phenomenon involving the deregulation of a variety of signaling pathways as a consequence of several genetic and epigenetic abnormalities. In this context, cumulative evidence supports a functional role of microRNAs in affecting radioresistance, suggesting the modulation of their expression as a novel radiosensitizing approach. Here, we investigated for the first time the ability of miR-205 to enhance the radiation response of PCa models.
Methods
miR-205 reconstitution by a miRNA mimic in PCa cell lines (DU145 and PC-3) was used to elucidate miR-205 biological role. Radiation response in miRNA-reconstituted and control cells was assessed by clonogenic assay, immunofluorescence-based detection of nuclear γ-H2AX foci and comet assay. RNAi was used to silence the miRNA targets PKCε or ZEB1. In addition, target-protection experiments were carried out using a custom oligonucleotide designed to physically disrupt the pairing between the miR-205 and PKCε. For in vivo experiments, xenografts generated in SCID mice by implanting DU145 cells stably expressing miR-205 were exposed to 5-Gy single dose irradiation using an image-guided animal micro-irradiator.
Results
miR-205 reconstitution was able to significantly enhance the radiation response of prostate cancer cell lines and xenografts through the impairment of radiation-induced DNA damage repair, as a consequence of PKCε and ZEB1 inhibition. Indeed, phenocopy experiments based on knock-down of either PKCε or ZEB1 reproduced miR-205 radiosensitizing effect, hence confirming a functional role of both targets in the process. At the molecular level, miR-205-induced suppression of PKCε counteracted radioresistance through the impairment of EGFR nuclear translocation and the consequent DNA-PK activation. Consistently, disruption of miR-205-PKCε 3’UTR pairing almost completely abrogated the radiosensitizing effect.
Conclusions
Our results uncovered the molecular and cellular mechanisms underlying the radiosensitizing effect of miR-205. These findings support the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa response to radiotherapy.
... In accordance with these results, we confirmed that Ars2 serves as an important mediator in regulating glioblastoma cell proliferation and maintaining stemness, and we suggest that Ars2 may be a potential therapeutic target for glioblastoma treatment. Moreover, Ars2 was reported to play important roles in microRNA biosynthesis (8,9,12), and consequently many microRNAs were found to affect cancer development in recent years (41)(42)(43)(44). It should be important to ascertain the association among Ars2, microRNAs and cancer. ...
It is generally known that glioblastoma is the most common primary malignant brain tumor and that it is highly aggressive and deadly. Although surgical and pharmacological therapies have made long-term progress, glioblastoma remains extremely lethal and has an uncommonly low survival rate. Therefore, further elucidation of the molecular mechanisms of glioblastoma initiation and its pathological processes are urgent. Arsenic resistance protein 2 (Ars2) is a highly conserved gene, and it has been found to play an important role in microRNA biosynthesis and cell proliferation in recent years. Furthermore, absence of Ars2 results in developmental death in Drosophila, zebrafish and mice. However, there are few studies on the role of Ars2 in regulating tumor development, and the mechanism of its action is mostly unknown. In the present study, we revealed that Ars2 is involved in glioblastoma proliferation and we identified a potential mechanistic role for it in cell cycle control. Our data demonstrated that Ars2 knockdown significantly repressed the proliferation and tumorigenesis abilities of glioblastoma cells in vitro and in vivo. Further investigation clarified that Ars2 deficiency inhibited the activation of the MAPK/ERK pathway, leading to cell cycle arrest in the G1 phase, resulting in suppression of cell proliferation. These findings support the conclusion that Ars2 is a key regulator of glioblastoma progression.
... We did not chose miR-125b in our studies, because miR-125b has been shown to behave as an oncomir under certain circumstances [42,43]. In contrast, both miR-125a and miR-205 are consistently found to act as tumor suppressors in breast cancers [44,45]. Indeed, our data showed that co-expression of miR-125a and miR-205 was more potent than either miRNA alone to inhibit erbB3 expression, decrease the levels of p-HER3, p-Src, p-Akt, and E2F1, and induce expression of p27 kip1 in HER2-overexpressing breast cancer cells. ...
Background
The HER3 receptor functions as a major cause of drug resistance in cancer treatment. It is believed that therapeutic targeting of HER3 is required to improve patient outcomes. It is not clear whether a novel strategy with two functional cooperative miRNAs would effectively inhibit erbB3 expression and potentiate the anti-proliferative/anti-survival effects of a HER2-targeted therapy (trastuzumab) and chemotherapy (paclitaxel) on HER2-overexpressing breast cancer cells.
Results
Combination of miR-125a and miR-205, as compared to either miRNA alone, potently inhibited expression of HER3 in HER2-overexpressing breast cancer BT474 cells. Co-expression of the two miRNAs not only reduced the levels of phosphorylated erbB3 (P-erbB3), Akt (P-Akt), and Src (P-Src), it also inhibited cell proliferation and increased cells at G1 phase. A multi-miRNA lentiviral vector - the cluster of miR-125a and miR-205 - was constructed to simultaneously express the two miRNAs in HER2-overexpressing breast cancer cells. Concurrent expression of miR-125a and miR-205 via the miRNA cluster transfection significantly enhanced trastuzumab-mediated growth inhibition and cell cycle G1 arrest in BT474 cells and markedly increased paclitaxel-induced apoptosis in another HER2-overexpressing breast cancer cell line HCC1954.
Conclusions
Here, we showed that functional cooperative miRNAs effectively suppressed erbB3 expression. This novel approach targeting of HER3 was able to enhance the therapeutic efficacy of trastuzumab and paclitaxel against HER2-overexpressing breast cancer.
Electronic supplementary material
The online version of this article (10.1186/s12575-018-0081-x) contains supplementary material, which is available to authorized users.
... In classical Hodgkin's lymphoma, plasma miR-21, miR-494 and miR-1973 are promising disease response biomarkers (9). miR-205 was identified previously as a target for inhibiting metastasis of breast cancer (10). A total of five members of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) are downregulated in tumor progression of breast cancer (11). ...
Bioinformatic tools were used to analyze GSE6188, GSE13937 and GSE43732 microarrays, and the top 10 upregulated and downregulated genes of each microarray were identified. It was determined that human microRNA (hsa-miR)-1 and hsa-miR-203 were two downregulated genes in common. Subsequently, it was identified that there were 145 and 335 genes in common targeted by hsa-miR-1 and hsa-miR-203, respectively. In order to narrow the number of target genes down further, the target genes were compared with GSE26886 microarray data. There were five upregulated genes in common with hsa-miR-1, i.e., MMD, BICD1, PTPRG, SDC2 and SEMA6D, and there were eight upregulated genes in common with hsa-miR-203, i.e., PXDN, NRCAM, FMNL2, EIF5A2, GLI3, FSL1, GREM1 and AHR. These genes may become promising biomarkers for the diagnosis of esophageal cancer.
... Gao et al. [25] demonstrated the possibility of the NW biosensor application for specific detection of lung cancer marker -miRNAs-126 -at 10 −16 М concentration. In the case of breast cancer, the literature discusses the following miRNAs as potential markers: miR1246, miR-4634, miR1307-3p, miR-6875-5p, miR-6861-5p, (miR)-10b [27,33]. ...
This work demonstrates the possibility of detection of DNA-oligonucleotide (oDNA) and microRNAs (miRNAs) extracted from blood plasma and associated with breast cancer (BC) in a buffer solution using the nanowire (NW) biosensor based on the silicon-on-insulator (SOI) structures. For biospecific detection, the NW surface was modified with oligonucleotide probes (oDNA-probes) complementary to the well-known miRNA sequence. It has been shown that the SOI-NW biosensor with immobilized oDNA-probes of this type can be used to detect complementary oDNA in a buffer solution with a concentration sensitivity of 10⁻¹⁷ М. The study also demonstrates the use of such biosensor for detection of an increased level of miRNAs isolated from the plasma of breast cancer patients. Application of the NW biosensor enables to distinguish between patients with a diagnosis of breast cancer from those with ovarian cancer (OC) by the value of an increased miRNAs level in the blood plasma of breast cancer patients.
... The diversity of expression of ErbB3 was related to its upstream regulation, among all this mechanism miRNA regulation was one of the important reasons. miR-125, miR-199a, miR-205, and miR-450 etc. were all reported to potentially capable of regulating ErbB3, generally by targeting its three prime untranslated regions (3′UTR) [5][6][7][8][9]. ...
Background/aims:
ErbB3 is an oncogene which has proliferation and metastasis promotion effects by several signaling pathways. However, the individual expression difference regulated by miRNA was almost still unknown. We focused on the miRNAs associated SNPs in the 3'-UTR of ErbB3 to investigate the further relationship of the SNPs with miRNAs among Chinese gastric cancer (GC) patients.
Methods:
We performed case-control study including 851 GC patients and 799 cancer-free controls. Genotyping, real-time PCR assay, cell transfection, the dual luciferase reporter assay, western-blot, cell proliferation and trans-well based cell invasion assay were used to investigate the effects of the SNP on ErbB3 expression. Moreover, a 5-years-overall survival and relapse free survival were investigated between different genotypes.
Results:
We found that patients suffering from Helicobacter pylori (Hp.) infection indicated to be the susceptible population by comparing with controls. Besides, SNP rs3202538 (G/T) in ErbB3 3'-UTR was involved in the occurrence of GC by acting as tumor risk factors. SNP rs3202538 (G/T) could be regulated by both miR-204 and miR-211 which caused an upregulation of ErbB3 in patients. Furthermore, the carriers of T genotype was related to the significantly high expression of ErbB3, and to big tumor size, poor differentiation as well as the high probability of metastasis. Both miR-211 and miR-204 can significantly decrease cell proliferation, metastasis as well as downstream AKT activation through G but not T allele of ErbB3 3'UTR. Moreover, the SNP of G/T was associated with shorter survival of post-surgery GC patients with 5 years of follow up study.
Conclusion:
In conclusion, our findings have shown that the SNP rs3202538 (G/T) in ErbB3 3'-UTR acted as promotion factors in the GC development through disrupting the regulatory role of miR-204 and miR-211 in ErbB3 expression.
... Oncogenic miRNA such as miR-21-5p [7,8,[41][42][43] are highly expressed in the cancer cell lines (MCF-7, MDA-MB-231, OE33, and SKGT4) and lowly expressed in the normal cell lines (HEEPIC and MCF10A). Conversely, the tumor suppressor miR-205-5p [41][42][43][44][45][46] is significantly overexpressed in the normal cell lines compared with the Determination of absolute expression profiles using multiplexed miRNA analysis cancer cell lines. However, we do not see reduced expression in the cancer cell lines for some other commonly reported tumor suppressors such as let-7a-5p [41][42][43]47], miR-15a-5p, and miR-16-5p. ...
Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR's ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes. © 2017 Song et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... Several deregulated miRNAs have been established in melanoma cells such as miR-26a and miR-15a [9,10] . In breast cancer, the miR-200 family has shown regulation of tumor progression and miR-205 inhibits metastasis [11,12] . Moreover, colorectal cancer metastasis and patient survival have been shown to be regulated by miR-184 and miR-133b [13] . ...
MiRNAs regulate gene expression post-transcriptionally and pre-translationally. Through gene regulation, several miRNAs have been found to play a significant role in various diseases. Each miRNA has multiple targets and is able to have a potent, albeit complex, effect on the cells. Specifically, miRNA-15a has been found to significantly reduce cancer cell survival and aggressiveness through multiple mechanisms across several cancer types. Our research found that miRNA-15a was able to decrease melanoma cell viability in-vitro and in-vivo. We have also found that miRNA-15a caused cell cycle arrest at the G0/G1 phase. Moreover, miRNA-15a was found to decrease the invasiveness of melanoma cells. CDCA4 was also discovered as a novel bona-fide target of miRNA-15a. The following oncogenic mRNAs are verified targets of miRNA-15a: CDCA4, BCL2L2, YAP1, AKT-3, Cyclin E1, and γ-Synuclein. In the future we hope to better understand which miRNAs will be effective in different transcriptome and genome environments. Efforts such as the NIH Center for Cancer Genomics' ‘The Cancer Genome Atlas,’ ‘Cancer Target and Driver Discovery Network,’ and the ‘Human Cancer Models Initiatives’ among others, will help us characterize the specific tumor environments in which different miRNAs are able to reduce cancer proliferation and aggression. This information will be enhanced by improving the delivery of miRNA by inducing its expression in-situ with dCas9 conjugated to activation domains.
... Furthermore, a miRNA may target multiple mRNA targets, and a target mRNA may be targeted by various miRNAs, which suggests that these regulatory RNAs exert complex, post-transcriptional control of gene expression (21,22). miRNAs have enormous potential to regulate various crucial biological processes, including the differentiation, progression, proliferation, apoptosis, metastasis and invasion of tumor cells (23)(24)(25)(26). ...
The aim of the present study was to evaluate the expression level of microRNA-182 (miRNA-182) in human osteosarcoma (OS) MG-63 cells and OS tissues, and to elucidate the effect of miRNA-182 on the biological activity of tumors. In the present study, the expression of miRNA-182 in human OS MG-63 cells, OS tissues and normal osteoblast hFOB1.19 cells was determined using quantitative polymerase chain reaction. Subsequently, a miRNA-182 mimic and inhibitor were utilized to regulate the expression level of this miRNA in MG-63 cells. Cell viability and proliferation were examined using cell counting kit-8 assays, and cell apoptosis was detected by flow cytometry. Cell invasion and migration assays were performed using Transwell chambers to analyze the biological functions of miRNA-182 in vitro. The present study demonstrated that the expression level of miRNA-182 in MG-63 cells and OS tissues was significantly increased compared with the hFOB1.19 cell line (P<0.05). The present study successfully performed cell transfections of miRNA-182 inhibitor and miRNA-182 mimic into MG-63 cells and achieved the desired transfection efficiency. The present study confirmed that upregulation of miRNA-182 promotes cell apoptosis and inhibits cell viability, proliferation, invasion and migration. The present findings additionally demonstrated that miRNA-182 is a tumor suppressor gene in OS. Therefore, regulating the expression of miRNA-182 may affect the biological behavior of OS cells, which suggests a potential role for miRNA-182 in molecular therapy for malignant tumors.
... Human miR-205 is located at the junction of the second intron and third exon of LOC642587 locus in chromosome 1. 66 It has been found that miR-205 is a highly conserved miRNA with homologs in different species. Recent studies on the functions of miR-205 have implicated its role in normal development and cancer. ...
Inflammatory breast cancer is the most aggressive form of breast cancer. Identifying new biomarkers to be used as therapeutic targets is in urgent need. Messenger RNA expression profiling studies have indicated that inflammatory breast cancer is a transcriptionally heterogeneous disease, and specific molecular targets for inflammatory breast cancer have not been well established. We performed microRNA expression profiling in inflammatory breast cancer in comparison with locally advanced noninflammatory breast cancer in this study. Although many microRNAs were differentially expressed between normal breast tissue and tumor tissue, most of them did not show differential expression between inflammatory and noninflammatory tumor samples. However, by microarray analysis, quantitative reverse transcription PCR, and in situ hybridization, we showed that microRNA-205 expression was decreased not only in tumor compared with normal breast tissue, but also in inflammatory breast cancer compared with noninflammatory breast cancer. Lower expression of microRNA-205 correlated with worse distant metastasis-free survival and overall survival in our cohort. A small-scale immunohistochemistry analysis showed coexistence of decreased microRNA-205 expression and decreased E-cadherin expression in some ductal tumors. MicroRNA-205 may serve as a therapeutic target in advanced breast cancer including inflammatory breast cancer.Modern Pathology advance online publication, 26 February 2016; doi:10.1038/modpathol.2016.38.
... A recent study showed that some miRNAs contribute to EMT induction (6). In addition, abnormal miRNA expression can lead to cancer and other diseases by suppressing the translation of the target gene's mRNA (7)(8)(9)(10)(11). OSCC-specific miRNAs have been identified (12), including miR-155. ...
Abnormal miRNA expression was recently implicated in the metastasis of oral squamous cell carcinoma (OSCC) and with a poor prognosis. The initiation of the invasion-metastasis cascade involves epithelial-mesenchymal transition (EMT). Our aim was to clarify how miRNA, especially miR-155-5p misexpression contributes to OSCC metastasis through EMT.
We collected tumor samples from 73 subjects with OSCC. The samples were analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and correlations between miR-155-5p levels and clinical characteristics were investigated. OSCC cell lines were analyzed by miRNA microarray and by transfection with a miR-155-5p mimic or inhibitor, followed by proliferation and wound-healing migration assays. qRT-PCR analyses of EMT makers in cells transfected with miR-155-5p inhibitor were performed.
We found high miR-155-5p expression in tissue samples from subjects with OSCC that had metastasized to cervical lymph nodes. HSC-3 cells also strongly expressed miR-155-5p. The epithelial marker E-cadherin was strongly expressed in HSC-3 cells transfected with miR-155-5p inhibitor, and we observed elevated SOCS1 and decreased STAT3 expression in these cells.
Our results suggest that miR-155-5p causes OSCC to metastasize, and could serve as a novel therapeutic target for OSCC.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
... For example, while miR-205 was overexpressed in endometrial cancer (54) and non-small cell lung cancer (55)(56)(57), its expression was downregulated in prostate cancer (58), melanoma (59)(60)(61), and breast cancers (62,63). The claimed tumor-suppressive functions of miR-205 in breast cancer is due to direct targeting of several oncogenes, such as VEGFA, E2F1, E2F5, PKC epsilon, and HER3 (64), as well as offsetting EMT by suppressing ZEB1 and ZEB2 (64)(65)(66). Reduced expression of miR-205 has been reported in with miR-222. Six groups of samples were listed. ...
Exposure to toxicants leads to cumulative molecular changes that over time increase a subject's risk of developing urothelial carcinoma (UC). To assess the impact of arsenic exposure at a time progressive manner, we developed and characterized a cell culture model and tested a panel of miRNAs in urine samples from arsenic exposed subjects, UC patients and controls. To prepare an in vitro model, we chronically exposed an immortalized normal human bladder cell line (HUC1) to arsenic. Growth of the HUC1 cells was increased in a time dependent manner after arsenic treatment and cellular morphology was changed. In soft agar assay, colonies were observed only in arsenic treated cells and the number of colonies gradually increased with longer periods of treatment. Similarly, invaded cells in invasion assay were observed only in arsenic treated cells. Withdrawal of arsenic treatment for 2.5 months did not reverse the tumorigenic properties of arsenic treated cells. Western blot analysis demonstrated decreased PTEN and increased AKT and mTOR in arsenic treated HUC1 cells. Levels of miR-200a, miR-200b, and miR-200c were down-regulated in arsenic exposed HUC1 cells by quantitative RT-PCR. Furthermore, in human urine, miR-200c and miR-205 were inversely associated with arsenic exposure (P=0.005 and 0.009, respectively). Expression of miR-205 discriminated cancer cases from controls with high sensitivity and specificity (AUC=0.845). Our study suggests that exposure to arsenic rapidly induces a multifaceted dedifferentiation program and miR-205 has potential to be used as a marker of arsenic exposure as well as a maker of early UC detection.
Copyright © 2015, American Association for Cancer Research.
... Similarly, some authors showed that high miR-185 or low miR-133b levels correlated with metastasis in colorectal cancer and high miR-155 or low miR-324a level in non-small cell lung carcinoma (NSCLC) could serve as prognostic indicators of such conditions. MiR-205 dysregulation and microRNA-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) downregulation has been proved in breast tumors (Gregory et al., 2008;Wu and Mo, 2009;Akcakaya et al., 2011). Accordingly, low miR-127 level was expressed in primary human bladder and prostate tumors in another study (Saito et al., 2006). ...
MiRNAs are endogenous, single stranded ~22-nucleotide non-coding RNAs (ncRNAs) which are transcribed by RNA polymerase II and mediate negative post-transcriptional gene regulation through binding to 3'untranslated regions (UTR), possibly open reading frames (ORFs) or 5'UTRs of target mRNAs. MiRNAs are involved in the normal physiology of eukaryotic cells, so dysregulation may be associated with diseases like cancer, and neurodegenerative, heart and other disorders. Among all cancers, lung cancer, with high incidence and mortality worldwide, is classified into two main groups: non-small cell lung cancer and small cell lung cancer. Recent promising studies suggest that gene expression profiles and miRNA signatures could be a useful step in a noninvasive, low-cost and repeatable screening process of lung cancer. Similarly, every stage of lung development during fetal life is associated with specific miRNAs. Since lung development and lung cancer phenomena share the same physiological, biological and molecular processes like cell proliferation, development and shared mRNA or expression regulation pathways, and according to data adopted from various studies, they may have partially shared miRNA signature. Thus, focusing on lung cancer in relation to lung development in miRNA studies might provide clues for lung cancer diagnosis and prognosis.
... Recent studies have targeted miR-205 for inhibiting the metastatic nature of BC. Wu et al [93] demonstrated that ectopic expression of the downregulated miR-205 effec-tively hinders cell proliferation, anchorage-independent growth and cell invasion, supporting its use as a possible therapeutic target. MiRNA delivery via nanoparticles is also a promising technique. ...
Breast cancer (BC) is the most frequent type of non skin cancer among women and a major leading cause of cancer-related deaths in Western countries. It is substantial to discover novel biomarkers with diagnostic, prognostic or predictive usefulness as well as therapeutic value for BC. Micro-RNAs (miRNAs) belong to a novel class of endogenous interfering RNAs that play a crucial role in post transcriptional gene silencing through mRNA targeting and, thus, are involved in many biological processes encompassing apoptosis, cell-cycle control, cell proliferation, DNA repair, immunity, metabolism, stress, aging, etc. MiRNAs exert their action mainly in a tumor suppressive or oncogenic manner. The specific aberrant expression patterns of miRNAs in BC that are detected with the use of high-throughput technologies reflect their key role in cancer initiation, progression, migration, invasion and metastasis. The detection of circulating extracellular miRNAs in plasma of BC patients may provide novel, non-invasive biomarkers in favor of BC diagnosis and prognosis and, at the same time, accumulating evidence has underscored the possible contribution of miRNAs as valuable biomarkers to predict response to chemotherapy or radiotherapy. Data from in vitro and in vivo studies on BC have revealed promising therapeutic approaches via miRNA delivery and miRNA inhibition. The purpose of this review is to explore the ontological role of miRNAs in BC etiopathogenesis as well as to highlight their potential, not only as non-invasive circulating biomarkers with diagnostic and prognostic significance, but also as treatment response predictors and therapeutic targets aiding BC management.
... Therefore miR-1 transfection and PTMA siRNA may have potential applications as an adjuvant in cancer chemotherapy. Although miR-205 was significantly reduced in breast tumors in which miR-205 act as the tumor suppressor through directly targeting of oncogenes such as ErbB3 and Zeb1 ( Wu & Mo, 2009;Wijnhoven et al., 2010), miR-205 has been shown to be more elevated in radio-resistant NPC cell line (CNE-2R) with suppressed PTEN protein expression than that in its parental cell line CNE-2. Knock-down miR-205 in CNE-2R cells compromised the inhibition of PTEN and increased cell apoptosis. ...
The definite molecular mechanisms underlying the genesis of nasopharyngeal carcinomas (NPCs) remain to be completely elucidated. miRNAs are small non-coding RNAs which are implicated in cell proliferation, apoptosis, and even carcinogenesis through negatively regulating gene expression post-transcriptionally. EBV was the first human virus found to express miRNAs. EBV-encoded BART-miRNAs and dysregulated cellular miRNAs are involved in carcinogenesis of NPC by interfering in the expression of viral and host cell genes related to immune responses and perturbing signal pathways of proliferation, apoptosis, invasion, metastasis and even radio-chemo-therapy sensitivity. Additional studies on the roles of EBV-encoded miRNAs and cellular miRNAs will provide new insights concerning the complicated gene regulated network and shed light on novel strategies for the diagnosis, therapy and prognosis of NPC.
Prostate cancer (PrCa) is one of the most common types of cancer among men in the United States. The metastatic and advanced PrCa develops drug resistance to current regimens which accounts for the poor management. microRNAs (miRNAs) have been well-documented for their diagnostic, prognostic, and therapeutic roles in various human cancers. Recent literature confirmed that microRNA-205 (miR-205) has been established as one of the tumor suppressors in PrCa. miR-205 regulates number of cellular functions, such as proliferation, invasion, migration/metastasis, and apoptosis. It is also evident that miR-205 can serve as a key biomarker in diagnostic, prognostic, and therapy of PrCa. Therefore, in this review, we will provide an overview of tumor suppressive role of miR-205 in PrCa. This work also outlines miR-205 specific role in targeted mechanisms for chemosensitization and radiosensitization in PrCa. A facile approach of delivery paths for successful translation is documented. Together, all these studies provide a novel insight of miR-205 as an adjuvant agent for reducing the widening gaps in clinical outcome of PrCa patients.
Ovine pulmonary adenocarcinoma (OPA) is an infectious neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus. OPA is an important veterinary problem and is also a valuable large animal model for human lung adenocarcinoma. JSRV infects type 2 alveolar epithelial cells in the lung and induces the growth of tumors, but little is known about the molecular events that lead to the activation of oncogenic pathways in infected cells. MicroRNAs (miRNAs) are small RNA molecules of approximately 22 nucleotides with important roles in regulating gene expression in eukaryotes and with well-established roles in cancer. Here we used small-RNA sequencing to investigate the changes in miRNA expression that occur in JSRV-infected ovine lung. After filtering out low abundance miRNAs, we identified expression of 405 miRNAs, 32 of which were differentially expressed in JSRV-infected lung compared to mock-inoculated control lung. Highly upregulated miRNAs included miR-182, miR-183, miR-96 and miR-135b, which have also been associated with oncogenic changes in human lung cancer. Network analysis of genes potentially targeted by the deregulated miRNAs identified their involvement in pathways known to be dysregulated in OPA. We found no evidence to support the existence of miRNAs encoded by JSRV. This study provides the first information on miRNA expression in OPA and identifies a number of targets for future studies into the role of these molecules in the pathogenesis of this unique veterinary model for human lung adenocarcinoma.
IMPORTANCE
Ovine pulmonary adenocarcinoma is a neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant welfare and economic concern for sheep producers and is a valuable large animal model for human lung adenocarcinoma. MicroRNAs are small RNA molecules of approximately 22 nucleotides with important functions in regulating gene expression in eukaryotes and with well-established roles in cancer. In this study, we examined the changes in microRNA expression that occur in the lung in response to JSRV infection. We identified differential expression of a number of host-encoded microRNAs in infected tissue, including microRNAs with roles in human cancer. We found no evidence that JSRV encodes a microRNA. This study provides new insights on the cellular response to JSRV infection in the ovine lung, which will inform future studies into the pathogenesis of OPA in sheep and its use as a model for human lung adenocarcinoma.
Nuclear factor I/B(NFIB) is a prominent transcription factor that plays a critical role in cancer progression. In this study, we found that the protein level of NFIB was significantly upregulated in estrogen receptor (ER) positive breast cancer tissues compared to matched adjacent noncancerous tissues while the NFIB mRNA expression level was not obviously dysregulated. Similarly, ER-positive breast cancer cell line, MCF7 express a high protein level of NFIB, while the mRNA level is not significantly upregulated. The function assays indicated that NFIB promoted MCF-7 cell cycle progression, cell proliferation and suppressed apoptosis in vitro. Furthermore, we explored the molecular mechanisms of NFIB as a target gene of miR-205-5p. Finally, we found that miR-205-5p was significantly downregulated in ER -positive breast cancer, and had the opposite eff ;ects on breast cancer cells compared with NFIB. Taken together, this study highlighted the molecular mechanisms of NFIB as an oncogene in ER-positive breast cancer, which was negatively regulated by miR-205-5p in breast cancer.
With the advent of genome-editing technologies, targeting genome engineering is no longer a hypothetical abstract. As application area of genome-editing tools is extending beyond the limits of research and biomedical remedies, specific ethical apprehensions are prevalent around the global community about the appropriate scope of genome-editing tools to be used. Genome-editing tools, i.e., meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats (CRISPR/Cas system), accelerate cancer research not only in its base study as well as in its cure by dissecting the mechanism of tumor development, categorizing targets for drug progression, and identifying arm cells for cell-dependent therapies. Current applications of cancer research and cure are discussed in this chapter. Moreover, it has also been discussed that genome editing is the possible cause of enhancing the risk of cancer development.
MicroRNAs also known as miRNAs/miRs are small, intrinsic non-coding RNAs having a vital role controlling the target gene expression. The significant role of microRNAs in tumours has been highlighted owing to different alterations such as amplification, deletion, point mutations in miRNA target genes, aberrant miRNA regulation at the transcriptional level, impaired epigenetic variations, dysregulation of the miRNA biogenesis mechanism as well as the complex interactions between miRNA and their target genes. These act either as oncogenes or tumour suppressors in certain conditions, and their dysregulation has been reported to influence different cancer hallmarks such as enduring proliferative signalling, escaping growth suppressors, opposing cell death and stimulating angiogenesis, tumour invasion as well as metastasis. Numerous reports have recognized miRNAs as impending biomarkers for diagnosis, prognosis of human tumours clinical assessment of patient outcome, disease monitoring and therapeutic targets and/or tools. Thus, this review encompasses the miRNAs regulation and mechanism that is involved in the advancement of human tumours and also sheds light on the clinical applications of miRNAs in different cancers.
Clinical manifestations of complex diseases like cancer oblige deep knowledge of each attribute. Both structural and functional aspects of the genome are essential for the characterization of DNA intended for sequencing the whole genome, accompanied with the knowledge of bioinformatics analysis and computational simulation, improving the selection of target biomarkers and drug designing strategies. In this chapter, we have given a brief outline of cancer genomics, sequencing methods (conventional and next-generation), biomarkers, DNA, and tissue microarrays. Recent investigations in different types of cancer demonstrate its relationship with gene mutations which assign the specific chromosome and physical mapping of genome structure using both experimental and computational models. This chapter discusses the cost-effectiveness and labor intensity of techniques utilized for gene manipulations which advance the practices for biomedical and clinical diagnostics strategies. On the other hand, biomarkers are the biological molecules which act as indicators for certain diseases and can be elevated or reduced during certain pathological conditions, consequently revealing the route to the detection, diagnosis, prognosis, and predictions for many disease classifications. Mass-spectrometric analysis of whole protein sequences is also getting attention due to its promising approaches, facilitating diagnosis in the field of infections as well as in the indication of pathological stages, with the help of protein release profiling and expression both qualitatively and quantitatively. DNA microarrays and tissue microarrays are the techniques that have been evolved for the economization of cost and time for both DNA and tissue analysis, respectively, minimizing the laborious procedures for diagnosis. The advantages of these aforementioned techniques continue to progress, thus, mesmerizing the scientific world.
Cancer proteomics is a diverse and challenging field. It provides the promising tool that aids in the understanding of the disease, yet early-stage diagnosis is still a question and crucial for successful treatment of cancer. Genomics aided with proteomics has emerged as a larger platform for understanding the disease spread, proliferation, metastasis, genomic aberrations, mutational changes, therapeutics, design drug delivery system, proteomic anomalies, structural changes, and signaling pathways and moving toward personalized approach. Biomarker identification and development of the panel are very precious in cancer treatment. Generally, biomarker identification, validation, and clinical examination are specific tools for accurate diagnostic, prognostic, and therapeutics. Present-day advances in proteomics and computational sciences have opened a gateway for the identification and quantitative analysis of protein variations associated with the complexities and heterogeneity of tumor development. Concept of personalized medicine is an emerging approach for cancer patient treatment, yet it has many challenges to overcome before its clinical application. Translational research in oncology still needs lots of quality research to overcome many challenges and for improvement in biomedical application and cancer patient care.
The development of novel therapies or the improvement of currently used approaches to treat prostate cancer (PCa), the most frequently diagnosed male tumor in developed countries, is an urgent need. In this regard, the functional characterization of microRNAs, molecules shown to regulate a number of cancer-related pathways, is instrumental to their possible clinical exploitation. Here, we demonstrate the tumor-suppressive role of the so far uncharacterized miR-1272, which we found to be significantly down-modulated in PCa clinical specimens compared to normal tissues. Through a gain-of-function approach using miRNA mimics, we showed that miR-1272 supplementation in two PCa cell models (DU145 and 22Rv1) reverted the mesenchymal phenotype by affecting migratory and invasive properties, and reduced cell growth in vitro and in vivo in SCID mice. Additionally, by targeting HIP1 encoding the endocytic protein HIP1, miR-1272 balanced EGFR membrane turnover, thus affecting the downstream AKT/ERK pathways, and, ultimately, increasing PCa cell response to ionizing radiation. Overall, our results show that miR-1272 reconstitution can affect several tumor traits, thus suggesting this approach as a potential novel therapeutic strategy to be pursued for PCa, with the multiple aim of reducing tumor growth, enhancing response to radiotherapy and limiting metastatic dissemination.
“Mediterranean diet and olive oil modify gene transcription towards a protective mode.”
There is emerging evidence that some microRNAs can promote or suppress several human cancer development and progression. However, the profile and molecular mechanism of microRNAs for human breast cancer is poorly unknown. We used bioinformatics approaches to find new candidate diagnostic and therapeutic miRNAs in human breast cancer via analysis of TCGA RNA sequencing data and publicly GEO microarray data, in order to provide theoretical basis for the future investigations of breast cancer. Decreased expression miR-146a was identified as a key regulator of human breast cancer development and progression. Interestingly, we founded that miR-146a expression levels dependent on tumor size and pathological grading in breast cancer patients, but not associated with other factors including age, T classification. Kaplan-Meier survival analysis showed that patients with high miR-146a expression had a longer survival rate than those low miR-146a expressions. In vitro assays of over-expression miR-146a induces cell cycle arrest and inhibits MDA-MB-231 cell proliferation. Furthermore, luciferase reporter gene assays demonstrated that miR-146a directly combine the 3-untranslated region of CDKN2A mRNA. In conclusion, we demonstrated miR-146a play an important role in breast cancer development and progression.
MicroRNAs (miRNAs) are a type of small non-coding RNA molecule that performs an important role in post-transcriptional gene regulation. Since miRNAs were first identified in 1993, a number of studies have demon-strated that they act as tumor suppressors or oncogenes in human cancer, including colorectal, lung, brain, breast and liver cancer, and leukemia. Large high-throughput studies have previously revealed that miRNA profiling is critical for the diagnosis and prognosis of patients with cancer, while certain miRNAs possess the potential to be used as diag-nostic and prognostic biomarkers or therapeutic targets in cancer. The present study reviews the studies and examines the roles of miRNAs in cancer diagnosis, prognosis and treatment, and discusses the potential therapeutic modality of exploiting miRNAs.
Increasing evidence has indicated that microRNAs (miRNAs) regulate gene expression at the post-transcriptional level. Aberrant miRNA expression has been associated with many types of human disease, including cancers. Their associations can be used to understand the pathogenesis of diseases. However, using experimental methods to identify the associations between diseases and miRNAs is time consuming and costly. Computational methods could find the most promising miRNA-disease associations in a short time, thereby significantly reducing experimental time and cost. This paper presents a network similarity integration method (NSIM) for predicting potential miRNA-disease associations, considering that diseases associated with highly related miRNAs are more similar (and vice versa). The NSIM is based on 5425 experimentally verified human miRNA-disease associations, which consist of 495 miRNAs and 381 diseases. The NSIM integrates the disease similarity network, miRNA similarity network, and known miRNA-disease association network on the basis of cousin similarity to predict novel miRNA-disease associations. We evaluate the NSIM using leave-one-out cross validation. The area under the curve of the method is 0.9475, indicating its outstanding performance. Case studies on prostate, breast, and colon neoplasms further proved the outstanding performance of the NSIM to predict not only disease-related miRNAs but also isolated diseases (diseases without any related miRNAs).
Ovarian cancer is one of the most common malignant tumor of female genital organs which ranks the third morbidity. We aimed to provide a better understanding of the mechanism of invasion and metastasis of ovarian cancer. The ovarian cancer samples were downloaded from GEO. Then clustering was performed to classify the stage of miRNAs based on the difference of prognosis and metastasis. Furthermore, the miRNAs model was build and the survival analysis processes was performed to observe the influence on prognosis, invasion and metastasis. At last, miRNAs co-expression network was built to explore the core miRNAs and the risk classification model was built to perform the risk assessment based on these core miRNAs. A total of 17 significantly differential expressed miRNAs were obtained. Functional enrichment of 1488 target genes, pathways like cell cycle, focal adhesion and pathways in cancer, which are closely related to the proliferation and metastasis of cancer cells were highly enriched, this indicate that these miRNAs are related to the proliferation and metastasis of cancer cells. The co-expressed network shows that the high expression of has-miR-320 indicated negative prognosis and high risk of metastasis. In conclusion, the expression level of has-miR-320 is highly related to the migration and invasion of cancer. The high expression of has-miR-320 directly indicated negative prognosis and high risk of metastasis. These findings reveal that hsa-miR-320 may serve as an important therapeutic target in ovarian cancer therapy. This article is protected by copyright. All rights reserved
Background
The expression of miR-205 is closely related to the occurrence, development, and prognosis of lung cancer and breast cancer. However, studies show that it plays opposite roles in different tumor types. Because the expression and regulation of miR-205 are primarily confined to epigenetic areas, whether genetic variation of miR-205 is related to the occurrence or to the development of tumors has not been reported. The aim of this study was to screen genetic variation of miR-205 gene and to investigate its association with the risk and development of lung and breast cancer.
Material/Methods
Genomic DNA was extracted from cultured tumor cell lines and formalin-fixed and paraffin-embedded lung and breast tissue samples. Bisulfite Clone Sequencing (BCS) and qRT-PCR were employed to detect the DNA methylation status and gene expression of the miR-205 gene, respectively. Genetic variation of miR-205 and miR-205HG were genotyped with PCR-sequencing method. Immunohistochemical analysis for ER, PR, and HER2 was performed on breast tissue samples.
Results
A polymorphism, rs3842530, located downstream of the miR-205 gene and in the fourth exon of the miR-205 host gene (miR-205HG), was screened. rs3842530 had no correlation with the risk of breast cancer, but was associated with the risk of lung cancer (P<0.05).
Conclusions
These results indicate that the functional association of rs3842530 in miR-205HG and lung cancer might provide a possible explanation for the tissue-dependent function of miR-205 in different tumors.
Breast cancer is the most common type of cancer in women whose prevalence is increasing every year. Common strategies for diagnosis, prognosis and specific treatment of breast cancer need improvements to increase patients’ survival. For this reason, there is growing number of efforts world-wide with molecular approaches. With the advent of microRNAs (miRNAs), they have been interested for almost all aspects of tumorgenesis and correlation of breast cancer and microRNAs was discovered for the first time in 2005. MiRNAs form a group of small noncoding RNAs which participate in regulation of gene expression and subsequently several biological processes and pathogenesis of various diseases. As other cancers, miRNAs involved in breast cancer are classified in two groups: the first group is tumor inducing miRNAs (also called oncomirs) that can induce tumor initiation and progression, and their expression is increased in cancerous cells. The second group is tumor suppressor miRNAs. In normal situation, tumor suppressor miRNAs prevent beginning and progression of breast cancer through suppressing the expression of various oncogenes. In this review we will give a general overview about miRNAs and breast cancer, and in the following, more discussion about tumor suppressor miRNAs, with focus on the best known of them and their targeted oncogenes and signaling pathways. Finally, we will point to application of this group of miRNAs in diagnosis, prognosis and treatment of patients.
Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0-2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells.
A variety of different somatic copy number alterations (SCNAs) have been reported in NF1-associated malignant peripheral nerve sheath tumours (MPNSTs). Indeed, SCNA analysis, based upon different platforms, is beginning to identify specific regulatory pathways associated with MPNSTs, thereby providing us with new potential targets for therapeutic intervention. Extensive studies have also been undertaken on gene expression in NF1-associated tumours but not yet in parallel with SCNA analysis. Although in some cancers, SCNAs are associated with the altered regulation of genes, an integrative study of SCNAs and gene expression is currently lacking in the context of NF1-associated tumours. It is to be expected that such an integrative study will aid in the identification not only of driver genes associated with NF1 tumourigenesis but also cancer-specific biological pathways and hence potential therapeutic targets. It is envisaged that a combination of next generation sequencing and powerful new bioinformatic tools will provide new insights into how high SCNA affects gene expression and the biological pathways. © 2012 Springer-Verlag Berlin Heidelberg. All rights are reserved.
Evidences have demonstrated key mediatory roles of microRNA-205 (miR-205) in normal physiology and its aberrant expression in many cancers. Indeed, miR-205 has been identified as both a tumour suppressive and oncogenic miRNA playing crucial roles in tumourigenesis through regulating different cellular pathways such as cell survival, apoptosis, angiogenesis and metastasis. As a tumour suppressor, miR-205 acts as an inhibitor of cell proliferation, migration and invasion. On the other hand, as an oncogene, miR-205 promotes tumour initiation and development. All these functions act through different target genes in various types of cancers. Also, miR-205 displays potential as a therapeutic target for different cancers. To conclude, miR-205 has important clinical and pathological correlations in different cancers and may act as a diagnostic and prognostic marker as well as new molecular target for cancer therapy.
MicroRNAs (miRNAs) are 21-23 nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs. miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation and apoptosis. Although strict tissue- and developmental-stage-specific expression is critical for appropriate miRNA function, mammalian transcription factors that regulate miRNAs have not yet been identified. The proto-oncogene c-MYC encodes a transcription factor that regulates cell proliferation, growth and apoptosis. Dysregulated expression or function of c-Myc is one of the most common abnormalities in human malignancy. Here we show that c-Myc activates expression of a cluster of six miRNAs on human chromosome 13. Chromatin immunoprecipation experiments show that c-Myc binds directly to this locus. The transcription factor E2F1 is an additional target of c-Myc that promotes cell cycle progression. We find that expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. These findings expand the known classes of transcripts within the c-Myc target gene network, and reveal a mechanism through which c-Myc simultaneously activates E2F1 transcription and limits its translation, allowing a tightly controlled proliferative signal.
Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of ~21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila , at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.
MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. Here, we systematically evaluate the minimal requirements for functional miRNA-target duplexes in vivo and distinguish classes of target sites with different functional properties. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. We present examples and genome-wide statistical support to show that both classes of sites are used in biologically relevant genes. We provide evidence that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families.
MicroRNAs (miRNAs) are regulators of myriad cellular events, but evidence for a single miRNA that can efficiently differentiate multipotent stem cells into a specific lineage or regulate direct reprogramming of cells into an alternative cell fate has been elusive. Here we show that miR-145 and miR-143 are co-transcribed in multipotent murine cardiac progenitors before becoming localized to smooth muscle cells, including neural crest stem-cell-derived vascular smooth muscle cells. miR-145 and miR-143 were direct transcriptional targets of serum response factor, myocardin and Nkx2-5 (NK2 transcription factor related, locus 5) and were downregulated in injured or atherosclerotic vessels containing proliferating, less differentiated smooth muscle cells. miR-145 was necessary for myocardin-induced reprogramming of adult fibroblasts into smooth muscle cells and sufficient to induce differentiation of multipotent neural crest stem cells into vascular smooth muscle. Furthermore, miR-145 and miR-143 cooperatively targeted a network of transcription factors, including Klf4 (Kruppel-like factor 4), myocardin and Elk-1 (ELK1, member of ETS oncogene family), to promote differentiation and repress proliferation of smooth muscle cells. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of smooth muscle cells.
An increasing amount of experimental evidence shows that microRNAs can have a causal role in breast cancer tumorigenesis as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate. HER2 overexpression is a hallmark of a particularly aggressive subset of breast tumors, and its activation is strictly dependent on the trans-interaction with other members of HER family; in particular, the activation of the PI3K/Akt survival pathway, so critically important in tumorigenesis, is predominantly driven through phosphorylation of the kinase-inactive member HER3. Here, we show that miR-205, down-modulated in breast tumors compared with normal breast tissue, directly targets HER3 receptor, and inhibits the activation of the downstream mediator Akt. The reintroduction of miR-205 in SKBr3 cells inhibits their clonogenic potential and increases the responsiveness to tyrosine-kinase inhibitors Gefitinib and Lapatinib, abrogating the HER3-mediated resistance and restoring a potent proapoptotic activity. Our data describe miR-205 as a new oncosuppressor gene in breast cancer, able to interfere with the proliferative pathway mediated by HER receptor family. Our study also provides experimental evidence suggesting that miR-205 can improve the responsiveness to specific anticancer therapies.
Limited information is available concerning the expression and role of microRNAs in prostate cancer. In this study, we investigated the involvement of miR-205 in prostate carcinogenesis. Significantly lower miR-205 expression levels were found in cancer than in normal prostate cell lines as well as in tumor compared with matched normal prostate tissues, with a particularly pronounced reduction in carcinomas from patients with local-regionally disseminated disease. Restoring the expression of miR-205 in prostate cancer cells resulted in cell rearrangements consistent with a mesenchymal-to-epithelial transition, such as up-regulation of E-cadherin and reduction of cell locomotion and invasion, and in the down-regulation of several oncogenes known to be involved in disease progression (i.e., interleukin 6, caveolin-1, EZH2). Our evidence suggests that these events are driven by the concurrent repression of specific predicted miR-205 targets, namely N-chimaerin, ErbB3, E2F1, E2F5, ZEB2, and protein kinase Cepsilon. Strikingly, the latter seemed to play a direct role in regulating epithelial-to-mesenchymal transition. In fact, its down-regulation led to a cell phenotype largely reminiscent of that of cells ectopically expressing miR-205. Overall, we showed for the first time that miR-205 exerts a tumor-suppressive effect in human prostate by counteracting epithelial-to-mesenchymal transition and reducing cell migration/invasion, at least in part through the down-regulation of protein kinase Cepsilon.
MicroRNAs (miRNAs) are endogenous, small, non-coding RNAs, which are capable of silencing gene expression at the post-transcriptional level. In this study, we report that miR-205 is significantly underexpressed in breast tumor compared to the matched normal breast tissue. Similarly, breast cancer cell lines, including MCF-7 and MDA-MB-231, express a lower level miR-205 than the non-malignant MCF-10A cells. Of interest, ectopic expression of miR-205 significantly inhibits cell proliferation and anchorage independent growth, as well as cell invasion. Furthermore, miR-205 was shown to suppress lung metastasis in an animal model. Finally, western blot combined with the luciferase reporter assays demonstrate that ErbB3 and vascular endothelial growth factor A (VEGF-A) are direct targets for miR-205, and this miR-205-mediated suppression is likely through the direct interaction with the putative miR-205 binding site in the 3'-untranslated region (3'-UTR) of ErbB3 and VEGF-A. Together, these results suggest that miR-205 is a tumor suppressor in breast cancer.
The tumor suppressor p53 negatively regulates a number of genes, including the proto-oncogene c-Myc, in addition to activating many other genes. One mechanism of the p53-mediated c-Myc repression may involve transcriptional regulation. However, it is not clear whether microRNAs (miRNAs) play a role in the p53-mediated posttranscriptional regulation of c-Myc. In this study, we show that a putative tumor suppressor, miR-145, is expressed through the phosphoinositide-3 kinase (PI-3K)/Akt and p53 pathways. Importantly, p53 transcriptionally induces the expression of miR-145 by interacting with a potential p53 response element (p53RE) in the miR-145 promoter. We further show that c-Myc is a direct target for miR-145. Although miR-145 silences the expression of c-Myc, anti-miR-145 enhances its expression. This specific silencing of c-Myc by miR-145 accounts at least in part for the miR-145-mediated inhibition of tumor cell growth both in vitro and in vivo. Finally, the blockade of miR-145 by anti-miR-145 is able to reverse the p53-mediated c-Myc repression. Together, these results define the role of miR-145 in the posttranscriptional regulation of c-Myc by p53 and suggest that, as a new member of the p53 regulatory network, miR-145 provides a direct link between p53 and c-Myc in this gene regulatory network.
MicroRNAs (miRNAs) are small, noncoding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also linked different sets of miRNAs to metastasis through either the promotion or suppression of this malignant process. Interestingly, epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is also emerging as a common hallmark of human tumors. Thus, we wondered whether there was a miRNA hypermethylation profile characteristic of human metastasis. We used a pharmacological and genomic approach to reveal this aberrant epigenetic silencing program by treating lymph node metastatic cancer cells with a DNA demethylating agent followed by hybridization to an expression microarray. Among the miRNAs that were reactivated upon drug treatment, miR-148a, miR-34b/c, and miR-9 were found to undergo specific hypermethylation-associated silencing in cancer cells compared with normal tissues. The reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumor growth, and inhibited metastasis formation in xenograft models, with an associated down-regulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6, and TGIF2. Most important, the involvement of miR-148a, miR-34b/c, and miR-9 hypermethylation in metastasis formation was also suggested in human primary malignancies (n = 207) because it was significantly associated with the appearance of lymph node metastasis. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of human cancer metastasis.
Abnormalities of the EGF receptor and/or the related ERBB2 receptor occur in a significant proportion of cases of human breast cancer and are important influences in the behaviour of this tumour type. In this report we demonstrate by nucleic acid analysis and immunohistochemistry that the recently recognised third member of this gene family, ERBB3, shows a wide range of expression in breast cancer, and shows stronger immunoreactivity than that observed in normal tissue in 43 out of 195 cases (22%) of primary breast cancer. Overexpression of ERBB3 appears to result from increased levels of gene transcription since in none of the cell lines or primary cancers analysed did we find evidence of gene amplification. High expression of ERBB3 is positively associated with the presence of lymph node metastases, but there was no demonstrable relationship with patient survival in this series.
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Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180erbB3, a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180erbB3 in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E4Y1)n (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180erbB3 autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180erbB3 is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180erbB3 possesses an impaired intrinsic tyrosine kinase activity.
Psoriatic skin is characterized by microvascular hyperpermeability and angioproliferation, but the mechanisms responsible are unknown. We report here that the hyperplastic epidermis of psoriatic skin expresses strikingly increased amounts of vascular permeability factor (VPF; vascular endothelial growth factor), a selective endothelial cell mitogen that enhances microvascular permeability. Moreover, two VPF receptors, kdr and flt-1, are overexpressed by papillary dermal microvascular endothelial cells. Transforming growth factor alpha (TGF-alpha), a cytokine that is also overexpressed in psoriatic epidermis, induced VPF gene expression by cultured epidermal keratinocytes. VPF secreted by TGF-alpha-stimulated keratinocytes was bioactive, as demonstrated by its mitogenic effect on dermal microvascular endothelial cells in vitro. Together, these findings suggest that TGF-alpha regulates VPF expression in psoriasis by an autocrine mechanism, leading to vascular hyperpermeability and angiogenesis. Similar mechanisms may operate in tumors and in healing skin wounds which also commonly express both VPF and TGF-alpha.
The clinical outcome is generally positive for patients with node-negative breast carcinoma (i.e., those who do not have detectable metastases in the lymph nodes) who have been treated with surgery or surgery plus radiation therapy. In about 30% of the patients, however, the disease recurs, and they are at risk of death. Determination of valid new prognostic indicators would improve the ability to identify patients at high risk of recurrence. Breast cancer can entail substantial development of new blood vessels within the tumor tissue, and it is known that the growth and metastasis of solid tumors are dependent on such angiogenesis. The conversion of tumor cells to an angiogenic phenotype may be preceded by a change in the balance of angiogenic growth factors and angiogenesis inhibitors.
This study was conducted to determine if the levels of vascular endothelial growth factor (VEGF) protein, a potent endothelial growth factor and mediator of vascular permeability and angiogenesis, measured in the primary tumors of women with node-negative breast cancer are associated with known prognostic factors and patient survival.
By use of a selective enzymatic immunoassay, levels of VEGF protein were measured in cytosolic extracts of primary tumor tissue surgically obtained from 260 women with node-negative breast carcinoma who had been treated with surgery with or without radiation therapy but not with adjuvant therapy and who had been followed for a median time of 66 months. The relationships between VEGF concentrations and other prognostic dichotomous variables or clinical outcome were tested by the use of the Kolmogorov-Smirnov test and univariate and multivariate Cox analyses, respectively. The relationship between VEGF and hormone receptors (i.e., those for estrogen and progesterone) was examined by the use of Spearman's correlation analyses. All P values resulted from the use of two-sided statistical tests.
Tumors from 247 (95%) of the 260 patients had detectable VEGF, ranging in concentration from 5.0 to 6523 pg/mg protein (median, 126.25 pg/mg protein). No statistically significant associations were found between VEGF and the other prognostic factors (e.g., age, menopausal status, histologic tumor type, tumor size, and hormone receptors) examined. Levels of VEGF were found to be prognostic for both relapse-free and overall survival in univariate and multivariate analyses (likelihood ratio tests; all four P values < .001). In the multivariate analysis, the first-order interaction term of VEGF and estrogen receptor was also prognostic for overall survival (likelihood ratio test; P = .05).
The results show that cytosolic levels of VEGF in tumor tissue samples are indicative of prognosis for patients with node-negative breast carcinoma.
The role of protein tyrosine kinase activity in ErbB3-mediated signal transduction was investigated. ErbB3 was phosphorylated in vivo in response to either heregulin (HRG) in cells expressing both ErbB3 and ErbB2, or epidermal growth factor (EGF) in cells expressing both ErbB3 and EGF receptor. A recombinant receptor protein (ErbB3-K/M, in which K/M stands for Lys-->Met amino acid substitution) containing an inactivating mutation in the putative ATP-binding site was also phosphorylated in response to HRG and EGF. Both the wild-type ErbB3 and mutant ErbB3-K/M proteins transduced signals to phosphatidylinositol 3-kinase, Shc and mitogen-activated protein kinases. Separate kinase-inactivating mutations in the EGF receptor and ErbB2 proteins abolished ErbB3 phosphorylation and signal transduction activated by EGF and HRG respectively. Hence the protein tyrosine kinase activity necessary for growth factor signalling via the ErbB3 protein seems to be provided by coexpressed EGF and ErbB2 receptor proteins.
Expression of c-erbB3 protein was investigated in 104 primary breast carcinomas comprising nine comedo ductal carcinoma in situ (DCIS), 91 invasive ductal carcinomas and four invasive lobular carcinomas using two monoclonal antibodies, RTJ1 and RTJ2. Of the 91 invasive ductal carcinomas, seven contained the comedo DCIS component adjacent to the invasive component. An immunohistochemical technique was used to evaluate the association between expression of c-erbB3 and clinical parameters and tumour markers such as epidermal growth factor receptor (EGFR), c-erbB2, cathepsin-D and p53 in archival formalin-fixed paraffin-embedded tumour tissues. Our results indicated that RTJ1 and RTJ2 gave identical staining patterns and concordant results. It was found that the overexpression of c-erbB3 protein was observed in 67% (6/9) of comedo DCIS, 52% (44/84) of invasive ductal carcinomas, 71% (5/7) of carcinomas containing both the in situ and invasive lesions and 25% (1/4) of invasive lobular carcinomas. A significant relationship (P < 0.05) was observed between strong immunoreactivity of c-erbB3 protein and histological grade, EGFR and cathepsin-D, but not with expression of c-erbB2, p53, oestrogen receptor status, lymph node metastases or age of patient. However, we noted that a high percentage of oestrogen receptor-negative tumours (59%), lymph node-positive tumours (63%) and c-erbB2 (63%) were strongly positive for c-erbB3 protein. We have also documented that a high percentage of EGFR (67%), c-erbB2 (67%), p53 (75%) and cathepsin-D-positive DCIS (60%) were strongly positive for c-erbB3. These observations suggest that overexpression of c-erbB3 protein could play an important role in tumour progression from non-invasive to invasive and, also, that it may have the potential to be used as a marker for poor prognosis of breast cancer.
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The discovery of numerous hypermethylated promoters of tumour-suppressor genes, along with a better understanding of gene-silencing mechanisms, has moved DNA methylation from obscurity to recognition as an alternative mechanism of tumour-suppressor inactivation in cancer. Epigenetic events can also facilitate genetic damage, as illustrated by the increased mutagenicity of 5-methylcytosine and the silencing of the MLH1 mismatch repair gene by DNA methylation in colorectal tumours. We review here current mechanistic understanding of the role of DNA methylation in malignant transformation, and suggest Knudson's two-hit hypothesis should now be expanded to include epigenetic mechanisms of gene inactivation.
To determine the predictive value of vascular endothelial growth factor (VEGF) for relapse-free survival (RFS) and overall survival (OS) in primary node-positive breast cancer (NPBC) after adjuvant endocrine treatment or adjuvant chemotherapy.
VEGF was quantitatively measured in tumor cytosols from 362 consecutive patients with primary NPBC using an enzyme immunoassay for human VEGF(165). Adjuvant treatment was given to all patients, either as endocrine therapy (n = 250) or chemotherapy (n = 112). The median follow-up time was 56 months.
Univariate analysis showed VEGF to be a significant predictor of RFS (P =.0289) and OS (P =.0004) in the total patient population and in patients who received adjuvant endocrine treatment (RFS, P =.0238; OS, P =.0121). In the group of patients who received adjuvant chemotherapy, no significant difference was seen in RFS, but a difference was seen in OS (P =.0235). Patients with bone recurrences tended to have lower VEGF expression (median, 2.17 pg/microg DNA) than patients with visceral metastasis (4.41 pg/microg), brain metastasis (8.29 pg/microg), or soft tissue recurrences (3.16 pg/microg). Multivariate analysis showed nodal status (P =.0004), estrogen receptor (ER) status (P <.0001), and tumor size (P =.0085) to be independent predictors of RFS. VEGF was found to be an independent predictor of OS (P =.0170; relative risk [RR] = 1.82), as were ER (P <.0001; RR = 5.19) and nodal status (P =.0002; RR = 2.58). For patients receiving adjuvant endocrine treatment, multivariate analysis showed VEGF content to be an independent predictor of OS (P =.0420; RR = 1.90) but not of RFS.
The results suggest that VEGF(165) content in tumor cytosols is a predictor of RFS and OS in primary NPBC. VEGF content might also predict outcome after adjuvant endocrine treatment, but further studies in a prospective setting with homologous treatments are required.
Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.
Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been reported to be associated with a poor prognosis in primary breast cancer and in several other cancer types. In the present study, we have measured with ELISA the levels of VEGF in cytosolic extracts of 845 primary breast tumors of patients who developed a recurrence during follow-up. All of the patients received tamoxifen (n = 618) or cyclophosphamide, methotrexate, 5-fluorouracil (CMF) or 5-fluorouracil, Adriamycin, cyclophosphamide (FAC) chemotherapy (n = 227) as first-line systemic therapy after diagnosis of advanced disease. VEGF levels were not related to age or menopausal status but were negatively related to the cytosolic levels of estrogen receptor and progesterone receptor (P < 0.0001). In patients who relapsed within 1 year after primary surgery, tumor VEGF levels were higher than in patients who showed a longer disease-free interval (P = 0.0005). In patients with a first relapse in the viscera, VEGF levels were higher compared with those that relapsed to the bone or soft tissue (P = 0.0004). In univariate analysis for response to first-line tamoxifen therapy, patients with high or intermediate levels showed a poor rate of response, compared with patients with low tumor-VEGF levels (P = 0.0001). Similarly, in multivariate analysis for response to tamoxifen treatment, corrected for age, site of relapse, disease-free interval, and estrogen receptor and progesterone receptor status, VEGF status was an independent predictive factor (P = 0.009). In concordance, higher levels of VEGF were associated with a short progression-free survival and postrelapse overall survival (both, P < 0.0001). On first-line chemotherapy, the rate of response decreased with higher tumor levels of VEGF, both in univariate (P = 0.003) and in multivariate analysis (P = 0.004). Furthermore, higher VEGF levels were associated with a short progression-free survival (P = 0.003) and postrelapse overall survival (P = 0.001). In conclusion, the tumor VEGF level is an important independent marker that predicts a poor efficacy of both tamoxifen and chemotherapy in advanced breast cancer. Knowledge of the tumor level of VEGF might be helpful in selecting individual patients who may benefit from treatments with antiangiogenic agents combined with conventionally used drugs.
Acute rejection is a major cause of reduced survival of renal allografts. Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and is expressed widely by renal tissue and T cells. VEGF influences adhesion and migration of leukocytes across the endothelium. This study investigates whether genetically determined variation in VEGF expression influences the development of renal allograft rejection. VEGF promoter polymorphisms were examined by using sequence-specific primer-PCR in 173 renal transplant recipients. Acute rejection occurred in 38.7%; median time to first rejection episode was 14 d. VEGF in vitro expression was investigated in stimulated leukocytes from 30 controls. The -1154*G and -2578*C alleles were associated with higher VEGF production. VEGF -1154 GG and GA genotypes were significantly associated with acute rejection risk at 3 mo (P = 0.004, odds ration [OR] = 6.8, 95% CI = 1.8 to 25 and P = 0.035, OR = 4.1, 95% CI = 1.1 to 15, respectively). Furthermore, VEGF -2578 CC and CA genotypes were associated with increased rejection risk (P = 0.005, OR = 4.1, 95% CI = 1.5 to 11.3 and P = 0.035, OR = 2.7, 95% CI = 1.1 to 7, respectively). These polymorphisms demonstrate linkage disequilibrium (P = 0.001). These data indicate that the -1154*G and -2578*C containing genotypes, encoding higher VEGF production, are strongly associated with acute rejection and may be useful markers of rejection risk.
bic is a novel gene identified at a common retroviral integration site in avian leukosis virus-induced lymphomas and has been
implicated as a collaborator with c-myc in B lymphomagenesis. It lacks an extensive open reading frame and is believed to function as an untranslated RNA (W. Tam,
Gene 274:157-167, 2001; W. Tam, D. Ben-Yehuda, and W. S. Hayward, Mol. Cell. Biol. 17:1490-1502, 1997). The oncogenic potential
of bic, particularly its ability to cooperate with c-myc in oncogenesis, was tested directly by expressing c-myc and bic, either singly or in pairwise combination, in cultured chicken embryo fibroblasts (CEFs) and in chickens using replication-competent
retrovirus vectors. Coexpression of c-myc and bic in CEFs caused growth enhancement of cells. Most importantly, chick oncogenicity assays demonstrated that bic can cooperate with c-myc in lymphomagenesis and erythroleukemogenesis. The present study provides direct evidence for the involvement of untranslated
RNAs in oncogenesis and provides further support for the role of noncoding RNAs as riboregulators.
Mutational inactivation of BRCA1 confers increased risk for breast cancer. However, the underlying basis for the breast tissue-restricted, tumor-suppressive properties of BRCA1 remains poorly defined. Here, we show that BRCA1 and the estrogen receptor alpha (ER-alpha) modulated vascular endothelial growth factor (VEGF) gene transcription and secretion in breast cancer cells. ER-alpha interacted in vitro and in vivo with BRCA1, and this interaction was mediated by the AF-2 domain of ER-alpha and two domains of BRCA1, the amino-acid residues 1-306 and 428-683. Endogenous interaction of ER-alpha with BRCA1 was observed in normal MCF-10A breast epithelial cells and in breast cancer cells (MCF-7 and T47D), and this interaction was significantly reduced in the presence of estrogen. Furthermore, ER-alpha induced activation of VEGF gene transcription, using human VEGF promoter-luciferase reporter constructs. The AF-2 domain of ER-alpha was also shown to induce VEGF gene transcription activation similar to that obtained with the full-length ER-alpha. However, in the presence of BRCA1, VEGF gene transcription activation and VEGF protein secretion were significantly inhibited in a dose-dependent manner. The BRCA1 domain of 1-683 amino acid residues was required for this inhibition of VEGF gene transcription activation. Three mutated forms of BRCA1 (A1708E, M1775R and Y1853X), that have been identified in familial breast cancers, failed to associate with ER-alpha and to suppress VEGF promoter activity and VEGF protein secretion. Overexpression of wild-type BRCA1 in HCC-1937 breast cancer cells that lack endogenous functional BRCA1 significantly reduced VEGF secretion in these cells. These results demonstrate a novel pathogenic mechanism whereby mutations in BRCA1, via their interaction with ER-alpha, could promote tumorigenesis through the hormonal regulation of mammary epithelial cell proliferation and impaired VEGF function, which may lead to cancer growth and angiogenesis.
Micro-RNAs (miR genes) are a large family of highly conserved noncoding genes thought to be involved in temporal and tissue-specific gene regulation. MiRs are transcribed as short hairpin precursors ( approximately 70 nt) and are processed into active 21- to 22-nt RNAs by Dicer, a ribonuclease that recognizes target mRNAs via base-pairing interactions. Here we show that miR15 and miR16 are located at chromosome 13q14, a region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL). Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority ( approximately 68%) of CLL cases.
Amyotrophic lateral sclerosis (ALS) is an incurable degenerative disorder of motoneurons. We recently reported that reduced expression of Vegfa causes ALS-like motoneuron degeneration in Vegfa(delta/delta) mice. In a meta-analysis of over 900 individuals from Sweden and over 1,000 individuals from Belgium and England, we now report that subjects homozygous with respect to the haplotypes -2,578A/-1,154A/-634G or -2,578A/-1,154G/-634G in the VEGF promoter/leader sequence had a 1.8 times greater risk of ALS (P = 0.00004). These 'at-risk' haplotypes lowered circulating VEGF levels in vivo and reduced VEGF gene transcription, IRES-mediated VEGF expression and translation of a novel large-VEGF isoform (L-VEGF) in vivo. Moreover, SOD1(G93A) mice crossbred with Vegfa(delta/delta) mice died earlier due to more severe motoneuron degeneration. Vegfa(delta/delta) mice were unusually susceptible to persistent paralysis after spinal cord ischemia, and treatment with Vegfa protected mice against ischemic motoneuron death. These findings indicate that VEGF is a modifier of motoneuron degeneration in human ALS and unveil a therapeutic potential of Vegfa for stressed motoneurons in mice.
ErbB2 is a receptor tyrosine kinase whose activity in normal cells depends on dimerization with another ligand-binding ErbB receptor. In contrast, amplification of c-erbB2 in tumors results in dramatic overexpression and constitutive activation of the receptor. Breast cancer cells overexpressing ErbB2 depend on its activity for proliferation, because treatment of these cells with ErbB2-specific antagonistic antibodies or kinase inhibitors blocks tumor cells in the G1 phase of the cell cycle. Intriguingly, loss of ErbB2 signaling is accompanied by a decrease in the phosphotyrosine content of ErbB3. On the basis of these results, it has been proposed that ErbB3 might be a partner for ErbB2 in promoting cellular transformation. To test this hypothesis and directly examine the role of the "kinase dead" ErbB3, we specifically ablated its expression with a designer transcription factor (E3). By infection of ErbB2-overexpressing breast cancer cells with a retrovirus expressing E3, we show that ErbB3 is an essential partner in the transformation process. Loss of functional ErbB2 or ErbB3 has similar effects on cell proliferation and cell cycle regulators. Furthermore, expression of constitutively active protein kinase B rescues the proliferative block induced as a consequence of loss of ErbB2 or ErbB3 signaling. These results demonstrate that ErbB2 overexpression and activity alone are insufficient to promote breast tumor cell division. Furthermore, we identify ErbB3's role, which is to couple active ErbB2 to the phosphatidylinositol 3-kinase/protein kinase B pathway. Thus, the ErbB2/ErbB3 dimer functions as an oncogenic unit to drive breast tumor cell proliferation.
MicroRNAs (miRNAs) are an abundant class of ∼22-nucleotide regulatory RNAs found in plants and animals. Some miRNAs of plants,
Caenorhabditis elegans, and Drosophila play important gene-regulatory roles during development by pairing to target mRNAs to specify posttranscriptional repression
of these messages. We identify three miRNAs that are specifically expressed in hematopoietic cells and show that their expression
is dynamically regulated during early hematopoiesis and lineage commitment. One of these miRNAs, miR-181, was preferentially
expressed in the B-lymphoid cells of mouse bone marrow, and its ectopic expression in hematopoietic stem/progenitor cells
led to an increased fraction of B-lineage cells in both tissue-culture differentiation assays and adult mice. Our results
indicate that microRNAs are components of the molecular circuitry that controls mouse hematopoiesis and suggest that other
microRNAs have similar regulatory roles during other facets of vertebrate development.
lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction.
Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.
The nematode worm Caenorhabditis elegans can be shy or gregarious when feeding time arrives. New work uncovers some of the neurons and genes that are involved in regulating social feeding in the worm, and points towards multiple systems of antagonistic signalling that control whether, and when, the worms aggregate into feeding groups.
Small RNA molecules have been known and utilized to suppress gene expression for more than a decade. The discovery that these small RNA molecules are endogenously expressed in many organisms and have a critical role in controlling gene expression has led to the arising of a whole new field of research. Termed small interfering RNA (siRNA) or microRNA (miRNA) these ∼ 22 nt RNA molecules have the capability to suppress gene expression through various mechanisms once they are incorporated in the multi-protein RNA-Induced Silencing Complex (RISC) and interact with their target mRNA. This review introduces siRNAs and microRNAs in a historical perspective and focuses on the key molecules in RISC, structural properties and mechanisms underlying the process of small RNA regulated post-transcriptional suppression of gene expression.
Thesis (Ph. D., Division of Medical Sciences (Genetics))--Harvard University, 1999. Includes bibliographical references.
MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression and play an important role in many developmental processes. We report here that expression of microRNA-145 (miR-145) is low in self-renewing human embryonic stem cells (hESCs) but highly upregulated during differentiation. We identify the pluripotency factors OCT4, SOX2, and KLF4 as direct targets of miR-145 and show that endogenous miR-145 represses the 3' untranslated regions of OCT4, SOX2, and KLF4. Increased miR-145 expression inhibits hESC self-renewal, represses expression of pluripotency genes, and induces lineage-restricted differentiation. Loss of miR-145 impairs differentiation and elevates OCT4, SOX2, and KLF4. Furthermore, we find that the miR-145 promoter is bound and repressed by OCT4 in hESCs. This work reveals a direct link between the core reprogramming factors and miR-145 and uncovers a double-negative feedback loop involving OCT4, SOX2, KLF4, and miR-145.
Recent advances in treatment of lung cancer require greater accuracy in the subclassification of non-small-cell lung cancer (NSCLC). Targeted therapies which inhibit tumor angiogenesis pose higher risk for adverse response in cases of squamous cell carcinoma. Interobserver variability and the lack of specific, standardized assays limit the current abilities to adequately stratify patients for such treatments. In this study, we set out to identify specific microRNA biomarkers for the identification of squamous cell carcinoma, and to use such markers for the development of a standardized assay.
High-throughput microarray was used to measure microRNA expression levels in 122 adenocarcinoma and squamous NSCLC samples. A quantitative real-time polymerase chain reaction (qRT-PCR) platform was used to verify findings in an independent set of 20 NSCLC formalin-fixed, paraffin-embedded (FFPE) samples, and to develop a diagnostic assay using an additional set of 27 NSCLC FFPE samples. The assay was validated using an independent blinded cohort consisting of 79 NSCLC FFPE samples.
We identified hsa-miR-205 as a highly specific marker for squamous cell lung carcinoma. A microRNA-based qRT-PCR assay that measures expression of hsa-miR-205 reached sensitivity of 96% and specificity of 90% in the identification of squamous cell lung carcinomas in an independent blinded validation set.
Hsa-miR-205 is a highly accurate marker for lung cancer of squamous histology. The standardized diagnostic assay presented here can provide highly accurate subclassification of NSCLC patients.
Small noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. The importance of miRNAs as potential cancer prognostic indicators is underscored by their involvement in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) tumor and adjacent normal tissue were examined by microarray analysis and validated by quantitative TaqMan real-time polymerase chain reaction. Using TaqMan real-time polymerase chain reaction we measured the quantitative associations between a subset of miRNAs identified on microarrays in primary tumors at diagnosis and cancer survival in a cohort of 104 HNSCC patients undergoing treatment with curative intent. The majority of miRNAs exhibiting altered expression in primary human HNSCC tumors (including miR-1, miR-133a, miR-205, and let-7d) show lower expression levels relative to normal adjacent tissue. In contrast, hsa-miR-21 is frequently overexpressed in human HNSCC tumors. Using univariate and multivariable statistical models we show that low levels of hsa-miR205 are significantly associated with loco-regional recurrence independent of disease severity at diagnosis and treatment. In addition, combined low levels of hsa-miR-205 and hsa-let-7d expression in HNSCC tumors are significantly associated with poor head and neck cancer survival Our results show that miRNA expression levels can be used as prognostic markers of head and neck cancer.
MicroRNAs (miRNAs) are small noncoding RNAs of 19-24 nucleotides in length and involved in gene expression regulation. They are associated with cell proliferation, differentiation, apoptosis, and carcinogenesis. The specific expression profiles of miRNAs have been found in many human cancers, but there are few studies on endometrioid adenocarcinoma. We found the miRNA expression profile in 10 pairs of endometrioid adenocarcinoma and adjacent nontumorous endometrium using human miRNA microarray. Seventeen miRNAs exhibited higher expression and six miRNAs exhibited lower expression in endometrioid adenocarcinoma samples than those in the nontumorous samples in the microarray. Of those, the miR-205, miR-449, and miR-429 were greatly enriched; in contrast the miR-204, miR-99b, and miR-193b were greatly downregulated in adenocarcinoma tissues. The expressions of these six miRNAs were validated using real time reverse transcription-PCR. This information may provide the candidate miRNA genome for further confirming the role of miRNAs in carcinogenesis of endometrioid adenocarcinoma and potentially serving as a diagnostic or therapeutic tool in endometrioid adenocarcinoma.
Despite their potential to regulate approximately one-third of the whole genome, relatively few microRNA (miRNA) targets have been experimentally validated, particularly in stratified squamous epithelia. Here we demonstrate not only that the lipid phosphatase SHIP2 is a target of miRNA-205 (miR-205) in epithelial cells, but, more importantly, that the corneal epithelial-specific miR-184 can interfere with the ability of miR-205 to suppress SHIP2 levels. This is the first example of a miRNA negatively regulating another to maintain levels of a target protein. Interfering with miR-205 function by using a synthetic antagomir, or by the ectopic expression of miR-184, leads to a coordinated damping of the Akt signaling pathway via SHIP2 induction. This was associated with a marked increase in keratinocyte apoptosis and cell death. Aggressive squamous cell carcinoma (SCC) cells exhibited elevated levels of miR-205. This was associated with a concomitant reduction in SHIP2 levels. Partial knockdown of endogenous miR-205 in SCCs markedly decreased phosphorylated Akt and phosphorylated BAD levels and increased apoptosis. We were able to increase SHIP2 levels in SCC cells after inhibition of miR-205. Therefore, miR-205 might have diagnostic value in determining the aggressivity of SCCs. Blockage of miR-205 activity with an antagomir or via ectopic expression of miR-184 could be novel therapeutic approaches for treating aggressive SCCs.
MicroRNAs (miRNAs), approximately 22-nt RNAs that mediate post-transcriptional regulation of mRNAs in animals and plants, are a diverse class of regulatory genes whose specific biological functions are largely unknown. Here we detail a protocol to design and introduce into cultured Drosophila and human cells sequence-specific antisense oligonucleotides (ASOs) that block the function of individual miRNAs. Coupled with recent studies that catalog the miRNAs expressed in diverse cultured cells, our method offers a rapid (<1 week) approach to validate miRNA targets and to study the cellular functions of individual human and Drosophila miRNAs. ASO-based inactivation of miRNAs is faster and simpler than comparable genetic or 'sponge'-based approaches, for which extensive recombinant DNA manipulation is required. We present our ASO design principles and an optimized transfection protocol in which transfection efficiency of Drosophila Schneider 2 cells can approach 100%. Our 3'-cholesterol-modified ASOs have enhanced potency, allowing miRNA inhibition for at least 7 d from a single transfection.
MicroRNAs have been implicated in regulating diverse cellular pathways. Although there is emerging evidence that some microRNAs can function as oncogenes or tumour suppressors, the role of microRNAs in mediating cancer metastasis remains unexplored. Here we show, using a combination of mouse and human cells, that microRNA-10b (miR-10b) is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion. Overexpression of miR-10b in otherwise non-metastatic breast tumours initiates robust invasion and metastasis. Expression of miR-10b is induced by the transcription factor Twist, which binds directly to the putative promoter of mir-10b (MIRN10B). The miR-10b induced by Twist proceeds to inhibit translation of the messenger RNA encoding homeobox D10, resulting in increased expression of a well-characterized pro-metastatic gene, RHOC. Significantly, the level of miR-10b expression in primary breast carcinomas correlates with clinical progression. These findings suggest the workings of an undescribed regulatory pathway, in which a pleiotropic transcription factor induces expression of a specific microRNA, which suppresses its direct target and in turn activates another pro-metastatic gene, leading to tumour cell invasion and metastasis.
Rapidly growing primary cultures of normal human mammary epithelial cells (HMEC) were exposed to 1 microgram of benzo[a]pyrene (B[a]P) per ml for two or three 24-hr periods. The B[a]P-treated populations consistently contained cells displaying a longer period of active growth in culture compared to the untreated control cells. Widespread heterogeneity in morphology and growth patterns was evidenced in these "extended life" (EL) cultures, with multiple sequential changes in these parameters occurring during the course of their life in culture. Two apparently immortal continuous cell lines have thus far emerged from these EL cultures. These lines have been characterized to be of human mammary epithelial origin and derived from the originally treated HMEC specimen. The continuous lines do not appear to be malignantly transformed as they do not cause tumor formation in nude mice and show little or no anchorage-independent growth. Nonetheless, they have acquired several properties characteristic of tumor-derived HMEC, which distinguish them from their normal progenitors. These cell lines, as well as the EL strains, may provide useful substrates for studies to determine what agents can induce further transforming events. Additionally, analysis of the multiple steps occurring in the El cultures, as well as in the emergence of the continuous cell lines, could potentially elucidate the processes occurring during human epithelial cell carcinogenesis and escape from senescence.
Solid tumors must induce a vascular stroma to grow beyond a minimal size, and the intensity of the angiogenic response has been correlated with prognosis in breast cancer patients. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a secreted protein that has been implicated in tumor-associated angiogenesis. Vascular permeability factor directly stimulates endothelial cell growth and also increases microvascular permeability, leading to the extravasation of plasma proteins, which alter the extracellular matrix in a manner that promotes angiogenesis. To determine whether VPF has a role in breast cancer, we used in situ hybridization to study VPF mRNA expression in normal breast tissue (13 specimens), comedo-type ductal carcinoma in situ (DCIS) (four specimens), infiltrating ductal carcinoma (12 specimens), infiltrating lobular carcinoma (two specimens), metastatic ductal carcinoma (three specimens) and metastatic lobular carcinoma (one specimen). Vascular permeability factor mRNA was expressed at a low level by normal duct epithelium but was expressed at high levels in tumor cells in all cases of comedo-type DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, VPF mRNA was not expressed at high levels in infiltrating lobular carcinoma. We also used in situ hybridization to study the expression of two recently described endothelial cell surface VPF receptors, flt-1 and kdr. Vascular permeability factor receptor mRNA was strongly expressed in endothelial cells of small vessels adjacent to malignant tumor cells in DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, no definite labeling for receptor mRNA was detected in infiltrating lobular carcinoma or nonmalignant breast tissue. The intense expression of VPF mRNA by breast carcinoma cells and of VPF receptor mRNA by endothelial cells of adjacent small blood vessels provides strong evidence linking VPF expression to the angiogenesis associated with comedo-type DCIS, infiltrating ductal, and metastatic ductal breast carcinoma.
lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction.
To determine the role of the two angiogenic peptides, vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP) (the latter also being a target enzyme for cytotoxicity of 5-fluorouracil and methotrexate), and conventional prognostic factors in predicting relapse-free survival (RFS) and overall survival (OS) probabilities in two cohorts of patients with node-positive breast cancer (NPBC) treated with either adjuvant chemotherapy (CMF [cyclophosphamide, methotrexate, 5-fluorouracil] schedule) or hormone therapy (tamoxifen).
We studied two groups of 137 and 164 patients with NPBC, median follow-up of 72 months for both, treated with adjuvant chemotherapy or hormone therapy, respectively. The cytosolic levels of VEGF and TP were determined in the primary tumor by original immunometric methods. The association between VEGF and TP and of these angiogenic peptides with other prognostic indicators were tested by using the Spearman correlation coefficient (for continuous variables) or the Kolmogorov-Smirnov test (for dichotomous variables). Results of the clinical outcome were analyzed by both univariate and multivariate (for RFS only) Cox regression models in which VEGF and TP were treated as continuous variables.
In the CMF group, the concentrations of VEGF and TP ranged from 5.8 to 7798 pg/mg of protein (median, 87.5 pg/mg) and from 1.2 to 904 U/mg (median, 138.2 U/mg), respectively. There was no significant association between the two angiogenic peptides. VEGF was not associated with any other variable, whereas TP showed a positive association with age and an inverse association with the number of involved nodes. In the tamoxifen group, the concentrations of VEGF (5.9-2482; median, 79.3 pg/mg protein) and TP (6.1-1542; median, 146.5 U/mg) were similar to those of the CMF group, and the two angiogenic peptides were not correlated. VEGF was positively associated with age and was inversely associated with estrogen receptor and progesterone receptor, whereas TP was not associated with any other variable. Univariate analysis in the CMF group showed that VEGF and TP were significantly predictive of both RFS and OS. Likewise, the number of involved axillary nodes was significantly associated with both RFS and OS. Univariate analysis in the tamoxifen group showed that TP did not significantly influence either RFS or OS. On the contrary, VEGF levels were significantly predictive of both RFS and OS, as were the number of involved nodes, estrogen receptor concentrations, and progesterone receptor concentration. In the multivariate analysis on RFS in the CMF group, VEGF, TP, their first-order interaction term, and age were significant and independent predictive factors. In the tamoxifen group, only VEGF and the number of involved nodes were significant and independent predictive factors.
The results of our study suggest that high levels of TP and low levels of VEGF characterize the patients with NPBC treated with adjuvant CMF who have the highest likelihood of favorable outcome. Low levels of VEGF and the presence of less than three involved axillary nodes characterize the patients with NPBC treated with adjuvant tamoxifen who have the highest likelihood of favorable outcome. This information may be useful to plan future studies to better select the patients with NPBC for conventional adjuvant treatments as well as to monitor the efficacy of novel therapeutic strategies of adjuvant therapy based on inhibition of angiogenesis.
The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.
Animal genomes contain an abundance of small genes that produce regulatory RNAs of about 22 nucleotides in length. These microRNAs are diverse in sequence and expression patterns, and are evolutionarily widespread, suggesting that they may participate in a wide range of genetic regulatory pathways.
Patterns of DNA methylation and chromatin structure are profoundly altered in neoplasia and include genome-wide losses of, and regional gains in, DNA methylation. The recent explosion in our knowledge of how chromatin organization modulates gene transcription has further highlighted the importance of epigenetic mechanisms in the initiation and progression of human cancer. These epigenetic changes -- in particular, aberrant promoter hypermethylation that is associated with inappropriate gene silencing -- affect virtually every step in tumour progression. In this review, we discuss these epigenetic events and the molecular alterations that might cause them and/or underlie altered gene expression in cancer.
We report that vascular endothelial growth factor (VEGF), a major angiogenic factor, is also arequisite autocrine factor for breast carcinoma invasion in vitro and that the VEGF receptor Neuropilin-1 but not Flt-1 is essential for this function. VEGF regulates expression of the chemokine receptor CXCR4, and this VEGF target is needed for invasion but not for cell survival. CXCR4 mediates migration of breast carcinoma cells toward stromal-derived factor-1, and this migration is dependent on autocrine VEGF. Of interest, a CXCR4-inhibitory peptide that is currently in HIV clinical trials suppressed invasion. Our findings indicate that a VEGF autocrine pathway induces chemokine receptor expression in breast carcinoma cells, thus promoting their directed migration toward specific chemokines.
MicroRNAs (miRNAs) are an abundant class of ∼22-nucleotide (nt) noncoding RNAs, some of which are known to control the expression of other genes at the posttranscriptional level ([1–4][1]). We developed a computational procedure (MiRscan) to identify miRNA genes ([5][2]) and apply it here to