No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
MicroRNA Expression Profiles Analysis of Apheresis Platelets Treated With Vitamin B2 and Ultraviolet-B During Storage
Abstract
Whether platelet (PLT) microRNA (miRNA) profiles are affected by pathogen reduction technology (PRT) using vitamin B2 and ultraviolet-B (VB2-PRT) remains unclear. Samples from VB2-PRT-treated (experimental group, E_) and untreated (control group, C_) apheresis PLTs were taken on days 1, 3 and 5 of storage, designated as E_1, E_3, E_5, C_1, C_3 and C_5, respectively. The miRNA expression profiles were assessed by DNA Nano Ball (DNB) sequencing technology, and verified by quantitive real-time fluorescence quantitative PCR (qRT-PCR). Compared with the expression profiles of PLT miRNAs, 3895 miRNAs were identified in the E_ groups while 4106 were in the C_ groups. There were 487 significant differentially expressed miRNAs in E_1 vs C_1 group, including 220 upregulated and 287 downregulated, such as miR-146a-5p and let-7b-5p. There were 908 significant differentially expressed miRNAs in E_3 vs C_3 group, including 297 upregulated and 611 downregulated, such as miR-142-5p and miR-7-5p. There were 229 significant differentially expressed miRNAs in E_5 vs C_5 group, including 80 upregulated and 149 downregulated, such as miR-3529-3p and miR-451a. These differentially expressed miRNAs had been suggested to have functional roles in energy homeostasis, cell communication, proliferation, migration and apoptosis. GO analysis showed a significant enrichmen in relevant biological process categories as receptor activity, signal transduction, cell transport, motility and chemotaxis. The significantly enriched KEGG pathway of predicted target genes was Glycosaminoglycan biosynthesis in E_ vs C_ groups. These new observation could provide insights on the understanding of change of miRNA profiles of PLT treated with VB2-PRT.
Ultraviolet (UV) is divided into UVA (long-wave, 320-400 nm), UVB (middle-wave, 280-320 nm) and UVC (short-wave, 100-280 nm) based on wavelength. UV radiation (UVR) from sunlight (UVA + UVB) is a major cause of skin photodamage including skin inflammation, aging and pigmentation. Accidental exposure to UVC burns the skin and induces skin cancer. In addition to the skin, UV radiation can also impair visual function. Non-coding RNAs (ncRNAs) are a class of functional RNAs that do not have coding activity but can control cellular processes at the post-transcriptional level, including microRNA (miRNA), long non-coding RNA (lncRNA) and circulatory RNA (circRNA). Through a review of the literature, it was determined that UVR can affect the expression of various ncRNAs, and that this regulation may be wavelength specific. Functionally, ncRNAs participate in the regulation of photodamage through various pathways and play pathogenic or protective regulatory roles. In addition, ncRNAs that are upregulated or downregulated by UVR can serve as biomarkers for UV-induced diseases, aiding in diagnosis and prognosis assessment. Therapeutic strategies targeting ncRNAs, including the use of natural drugs and their extracts, have shown protective effects against UV-induced photodamage. In the present review, an extensive summarization of previous studies was performed and the role and mechanism of ncRNAs in UV-induced radiation effects was reviewed to aid in the diagnosis and treatment of UV-related diseases.
The liver plays a critical role in metabolic processes, making it vulnerable to injury. Researchers often study carbon tetrachloride (CCl4)-induced hepatotoxicity in model organisms because it closely resembles human liver damage. This toxicity occurs due to the activation of various cytochromes, including CYP2E1, CYP2B1, CYP2B2, and possibly CYP3A, which produce the trichloromethyl radical (CCl3*). CCl3* can attach to biological molecules such as lipids, proteins, and nucleic acids, impairing lipid metabolism and leading to fatty degeneration. It can also combine with DNA to initiate hepatic carcinogenesis. When exposed to oxygen, CCl3* generates more reactive CCl3OO*, which leads to lipid peroxidation and membrane damage. At the molecular level, CCl4 induces the release of several inflammatory cytokines, including TNF-α and NO, which can either help or harm hepatotoxicity through cellular apoptosis. TGF-β contributes to fibrogenesis, while IL-6 and IL-10 aid in recovery by minimizing anti-apoptotic activity and directing cells toward regeneration. To prevent liver damage, different interventions can be employed, such as antioxidants, mitogenic agents, and the maintenance of calcium sequestration. Drugs that prevent CCl4-induced cytotoxicity and proliferation or enhance CYP450 activity may offer a protective response against hepatic carcinoma.
The epigenetic modifications play a vital role in gene regulation associated with different pathologies. Various nutrients in our diet, such as vitamins, can modulate these epigenetic mechanism.They also can regulatenderlying pathophysiological factors and processes that directly or indirectly. Most importantly, A, B, C, and D vitamins have recently been shown to be involved in this type of regulation together with vitamins E and K. Despite their effect on the DNA methylation process, an in-depth understanding of vitamin-mediated epigenetic alterations have yet to be investigated. Moreover, the role of vitamins in DNA methylation as nutraceuticals might be important to use for targeted therapy of various human diseases. Overall, this review provides a brief survey of the role of vitamins as epigenetic modulators or nutraceuticals, emphasizing their potential in epigenetic therapy.
Background/aims:
Emerging evidence suggests that exosomal microRNAs (miRNAs) mediate hepatoma progression through the post-translational regulation of their targets. However, characteristically-expressed miRNAs and their functions in the tumor and tumor-associated angiogenesis remain poorly understood.
Methods:
miRNA sequencing (HiSeq 2500 SE50) was performed to identify miRNA species that are involved in the hepatocellular carcinoma (HCC) pathogenesis. We identified miR-451a downregulation according to its expression and TCGA analysis. miR-451a was found to be mainly involved in cell viability, apoptosis, cell cycle and migration both in HCC and endothelial cell lines. LPIN1 was predicted to be a target of this miRNA based on TargetScan, GSEA analysis, and the Uniprot database. We performed real time PCR and dual luciferase assays to confirm these results.
Results:
We identified that miR-451a is significantly downregulated in serum-derived exosomes from HCC patients, as compared to expression in those from normal individuals. We further confirmed that overexpression of miR-451a functions in HCC and endothelia cells in vitro and in vivo. Exosomal miR-451a, as a tumor suppressor, was found to induce apoptosis both in HCC cell lines and human umbilical vein endothelial cells (HUVECs). In addition, miR-451a suppressed HUVEC migration, tube formation, and vascular permeability. Importantly, we demonstrated that LPIN1 is a critical target of miR-451a, and promotes apoptosis in both HCC and endothelial cells.
Conclusion:
Our study provides the novel finding that exosomal miR-451a targets LPIN1 to inhibit hepatocellular tumorigenesis by regulating tumor cell apoptosis and angiogenesis. These results have clinical implications regarding the deregulation of miRNAs in HCC.
The bacterial keratitis causes viability loss and apoptosis in the corneal epithelial cells (CECs). The cyanidin-3-O-glucoside (C3G) benefits visual system and also possess anti-bacterial and anti-inflammatory potentials. In the current study, the effects of C3G on human CECs (HCECs) against bacterial lipopolysaccharide (LPS)-induced disorders were assessed, and the mechanism driving the protective effect was explored by focusing on let-7b-5p-mediated HMGA2/PI3K/Akt pathway. The HCECs were incubated LPS of P. aeruginosa to induce inflammation and apoptosis, and then treated with C3G. The changes in cell viability, apoptosis, and inflammation were detected. Moreover, the effects of LPS and C3G on let-7b-5p level and HMGA2/PI3K/Akt pathway activity were also assessed. Thereafter, the HCECs were further transfected with let-7b-5p inhibitor to confirm its role in the vision-protective effects of C3G. The interaction between let-7b-5p and HMGA2 was verified with dual luciferase assay. The LPS treatment suppressed viability and induced apoptosis and inflammation in HCECs, which was associated with the down-regulated let-7b-5p level and up-regulated HMGA2/PI3K/Akt pathway activity. The impairments of LPS on HCECs were attenuated by C3G: the compound increased cell viability and inhibited apoptosis and inflammation. The C3G also induced let-7b-5p level and inactivated HMGA2/PI3K/Akt pathway. However, after the inhibition of let-7b-5p, the protective effects of C3G on HCECs against LPS were blocked. The results of dual luciferase assay showed the direct binding let-7b-5p to the promoter of HMGA2 gene. It was inferred that the C3G could ameliorate the LPS-induced disorders in HCECs. The effect depended on the induced level of let-7b-5p, which then inhibited HMGA2/PI3K/Akt pathway.
Despite improvements in donor screening and increasing efforts to avoid contamination and the spread of pathogens in clinical platelet concentrates (PCs), the risks of transfusion-transmitted infections remain important. Relying on an ultraviolet photo activation system, pathogen reduction technologies (PRTs), such as Intercept and Mirasol, utilize amotosalen, and riboflavin (vitamin B2), respectively, to mediate inactivation of pathogen nucleic acids. Although they are expected to increase the safety and prolong the shelf life of clinical PCs, these PRTs might affect the quality and function of platelets, as recently reported. Upon activation, platelets release microparticles (MPs), which are involved in intercellular communications and regulation of gene expression, thereby mediating critical cellular functions. Here, we have used small RNA sequencing (RNA-Seq) to document the effect of PRT treatment on the microRNA profiles of platelets and derived MPs. PRT treatment did not affect the microRNA profile of platelets. However, we observed a specific loading of certain microRNAs into platelet MPs, which was impaired by treatment with Intercept or its Additive solution (SSP+). Whereas, Intercept had an impact on the microRNA profile of platelet-derived MPs, Mirasol did not impact the microRNA profile of platelets and derived MPs, compared to non-treated control. Considering that platelet MPs are able to transfer their microRNA content to recipient cells, and that this content may exert biological activities, those findings suggest that PRT treatment of clinical PCs may modify the bioactivity of the platelets and MPs to be transfused and argue for further investigations into PRT-induced changes in clinical PC content and function.
Background
Numerous researches have demonstrated that miR-142-5p plays significant roles in several cancers, although the functional characteristic of miR-142-5p in breast cancer has not been determined. This study is designed to explore the biological significance of miR-142-5p in breast cancer clinical implication and mechanism of action.
Methods
The differential expression patterns of miR-142-5p and Sorbin and SH3 domain-containing protein 1 and correlations between them and clinical significances were analyzed based on data from database. The expression levels of miR-142-5p in breast cancer cells were detected using quantitative real-time polymerase chain reaction. Cell counting kit-8, transwell, and wound healing assays were used to explore the potential functions of miR-142-5p in breast cancer cells. In addition, bioinformatics prediction analysis and luciferase reporter assay were utilized to predict and identify the potential target gene of miR-142-5p. A rescue experiment was conducted by transfecting miR-142-5p inhibitors and si-Sorbin and SH3 domain-containing protein 1 into cells to explore miR-142-5p/Sorbin and SH3 domain-containing protein 1 pairs on breast cancer cells behaviors.
Results
The analysis results showed that miR-142-5p was highly expressed in patients with breast cancer, while Sorbin and SH3 domain-containing protein 1 presented a trend of low expression. The clinical significances analysis suggested that the overexpression of miR-142-5p is closely correlated with metastasis, while low expression of Sorbin and SH3 domain-containing protein 1 is correlated with clinicopathological characteristics and poor overall survival in patients with breast cancer. In vitro exploration, the expression of miR-142-5p was upregulated in breast cancer cells and inhibition of miR-142-5p expression significantly reduced the proliferation, invasion, and migration of breast cancer cells. Through rescue experiments, breast cancer cells proliferation, invasion, and migration reduction induced by silencing of miR-142-5p were reversed via knockdown Sorbin and SH3 domain-containing protein 1.
Conclusion
Our findings insinuate that miR-142-5p functions as a positive regulator of promoting breast cancer cells biological behaviors and clinical metastasis, possibly regulated by targeting Sorbin and SH3 domain-containing protein 1, thus providing valuable information in the development of preventive or even therapeutic strategies for utilizing miR-142-5p as a promising target.
MicroRNAs are known to be critical regulators in tumor progression. miR‐7‐5p was reported to be involved in several cancers, including glioblastoma, cervical cancer and melanoma but, its prognostic value and biological function in non‐small cell lung cancer (NSCLC) remains unclear. In this study, using quantitative real time PCR analysis, we found that miR‐7‐5p was significantly down‐regulated in NSCLC tissues and cell lines. Lower miR‐7‐5p expression was associated with TNM stage and tumor size by Chi‐squared test. Deceased miR‐7‐5p expression was associated with a worse prognosis in patients with NSCLC using Kaplan‐Meier survival analysis and Multivariate Cox regression analysis. Experiments in NSCLC cell lines (A549 and H1299) demonstrated that up‐regulation of miR‐7‐5p significantly suppressed cell proliferation, but induced cell cycle G0/G1 phase arrest and apoptosis using CCK‐8, colony formation and flow cytometry analysis. Through loss‐of‐function assays, we further demonstrated that down‐regulation of miR‐7‐5p promoted cell proliferation, cell cycle G1/S transition, but decreased cell apoptosis in SPC‐A1 cells. Furthermore, P21‐activated kinase 2 (PAK2) was predicted and confirmed as a direct target gene of miR‐7‐5p in NSCLC cells by luciferase reporter assay. In addition, we found PAK2 overexpression could partially reverse the effects of miR‐7‐5p on cell proliferation, cell cycle distribution and apoptosis. We thus concluded that lower expression of miR‐7‐5p was associated with poor prognosis and NSCLC progression by directly targeting PAK2.
MicroRNAs are a class of noncoding RNAs that can be involved in the regulation of gene expression in cancers, including lung cancer. Our previous research has shown that miR-486-5p is one of the most downregulated microRNAs in tissue and serum samples of lung cancer as a good diagnostic biomarker. The objective of this study is to investigate the roles of miR-486-5p in the progression of lung cancer. In this study, miR-486-5p was further validated to be significantly downregulated in additional nonsmall cell lung cancer (NSCLC) tissue, serum, and cell samples by quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the expression level of miR-486-5p was significantly associated with clinical phenotype of NSCLC. The PIK3R1 gene was confirmed to be a direct target of miR-486-5p by dual-luciferase reporter assay, and the expression level of miR-486-5p was inversely correlated with that of PIK3R1 in tumor tissues (r = −0.774, p < 0.01). Overexpressed miR-486-5p effectively inhibited cell proliferation and invasion and successfully induced apoptosis in vitro. PIK3R1 was involved in the suppression of miR-486-5p on cell growth. It can be concluded that miR-486-5p may act as a tumor suppressor contributing to the progression of NSCLC, and miR-486-5p would be a diagnostic and prognostic biomarker and a potential therapeutic target for lung cancer.
Background
Colorectal cancer (CRC) is one of the most common tumors, the causes of which remain unclear. Recently, many kinds of long non-coding RNAs (lncRNAs) have been identified to have an important role in the biological function of CRC. However, the effect of lncRNA zinc finger antisense 1 (ZFAS1) on development of CRC is still incompletely clear.
Material/Methods
Firstly, the expression of ZFAS1 and microRNA (miR)-7-5p in 40 CRC tissues and adjacent tissues was measured by real-time polymerase chain reaction. Then, we detected the cell proliferation, migration, invasion, and apoptosis in CRC cell lines by using Cell Counting Kit-8 assay, colony formation assay, flow analysis, and Transwell assay, respectively. Then, the relationship between ZFAS1 and miR-7-5p was verified by luciferase reporter assay. Finally, rescue experiments were conducted to confirmed that interaction of ZFAS1 and miR-7-5p in vitro.
Results
Our results showed that ZFAS1 was upregulated in CRC tissues, correlated with overall survival rates, and negatively related to the expression of miR-7-5p. It was verified that miR-7-5p was a direct target of ZFAS1 by bioinformatics analysis and luciferase reporter assay. In addition, knockdown of miR-7-5p inhibited proliferation, migration, and invasion, and promoted apoptosis in CRC cell lines, which could be rescue by miR-7-5p inhibitor.
Conclusions
Our study indicated that ZFAS1 directly targeted miR-7-5p, and knockdown of it could inhibit tumor growth, migration, invasion, and induce apoptosis in CRC. These data might provide a potent treatment mechanism or promising biomarker for CRC.
Tumor-associated lymphatic vessels (LV) serve as a route of cancer dissemination through the prometastatic crosstalk between lymphatic endothelial cells (LECs) lining the LVs and cancer cells. Compared to blood endothelial cell-derived angiocrine factors, however, LEC-secreted factors in the tumor microenvironment and their roles in tumor metastasis are poorly understood. Here, we report that ELK3 expressed in LECs contributes to the dissemination of cancer cells during tumor growth by providing oncogenic miRNAs to tumor cells through exosomes. We found that conditioned medium from ELK3-suppressed LECs (LCM) lost its ability to promote the migration and invasion of breast cancer cells such as MDA-MB-231, Hs578T and BT20 in vitro. Suppression of ELK3 in LECs diminished the ability of LECs to promote tumor growth and metastasis of MDA-MB-231 in vivo. Exosomes derived from LECs significantly increased the migration and invasion of MDA-MB-231 in vitro, but ELK3 suppression significantly diminished the pro-oncogenic activity of exosomes from LECs. Based on the miRNA expression profiles of LECs and functional analysis, we identified miR-503-3p, miR-4269 and miR-30e-3p as downstream targets of ELK3 in LECs, which cause the above phenotype of cancer cells. These findings strongly suggest that ELK3 expressed in LECs is a major regulator that controls the communication between the tumor microenvironment and tumors to support cancer metastasis.
Background/aims:
microRNA-486-5p (miR-486-5p) and forkhead box protein O1 (FOXO1) play an important role in the development of intervertebral disc degeneration (IDD). However, their molecular mechanisms in IDD remain unknown.
Methods:
qRT-PCR assay was used to identify miR-486-5p expression in nucleus pulposus (NP) cells. In-vitro transfection, CCK-8, flow cytometry and luciferase reporter assay were used to validate the role and relationship of miR-486-5p and FOXO1 in lipopolysaccharides (LPS)-stimulated NP cells. qRT-PCR and Western blot were used to measure the expression levels of inflammatory cytokines, matrix degrading enzymes, and extracellular matrix (ECM)-related genes.
Results:
miR-486-5p expression was significantly down-regulated, while FOXO1 expression was up-regulated in LPS-treated NP cells (P<0.001). miR-486-5p over-expression repressed LPS-induced expressions of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and matrix degrading enzymes (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5), and promoted the expressions of LPS-inhibited ECM-related genes (Aggrecan and Collagen II) (all P<0.001). In addition, miR-486-5p over-expression protected NP cells against LPS-induced apoptosis. However, inhibition of miR-486-5p led to the opposite effects. Mechanically, FOXO1 was a direct target gene of miR-486-5p. Over-expressed FOXO1 aggravated LPS-induced injury, and antagonized protection effects of miR-486-5p.
Conclusion:
miR-486-5p can inhibit inflammatory response, ECM degradation and apoptosis in NP cells by directly targeting FOXO1, which may contribute to the biological therapy of IDD.
Background
This study aimed to investigate whether miR‐146a‐5p was involved in the pathogenesis of thoracic aortic dissection (AD) via regulating the biological function of vascular smooth muscle cells (VSMCs).
Methods
Circulating miR‐146a‐5p level was measured by quantitative polymerase chain reaction (qPCR) in AD patients and healthy controls. Human dissected aortic samples were obtained from patients with thoracic AD Stanford type A undergoing surgical repair, and normal control samples were from organ donors who died from nonvascular diseases. The expression level of miR‐146a‐5p was detected using qPCR in each sample. The expression of SMAD4, which is involved in the TGF‐β pathway and indicated as the target gene of miR‐146a‐5p, was measured by qPCR and Western blot analysis at the mRNA level and protein level, respectively. Subsequently, VSMCs were transfected with miR‐146a‐5p mimics or inhibitors in vitro. VSMC proliferation and migration were detected using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide and Transwell assay, respectively. Flow cytometry was used to identify apoptosis. The expression of SMAD4 in VSMCs was determined using qPCR and Western blot analysis.
Results
Plasma level of miR‐146a‐5p is significantly higher in the AD group as compared with the control group. The expression of miR‐146a‐5p was significantly upregulated in dissected aorta compared with controls (P < 0.05). The overexpression of miR‐146a‐5p significantly induced VSMC proliferation and migration in vitro.
Conclusions
The expression of SMAD4 was modulated by miR‐146a‐5p. miR‐146a‐5p induced VSMC proliferation and migration through targeting SMAD4 and hence might be potentially involved in the development of AD.
The global morbidity and mortality of colorectal cancer (CRC) are ranked the third among gastrointestinal tumors in the world. MiR-451a is associated with several types of cancer, including CRC. However, the roles and mechanisms of miR-451a in CRC have not been elucidated. BAP31 is a predicted target gene of miR-451a in our suppression subtractive hybridization library. Its relationship with miR-451a and function in CRC are unclear. We hypothesized that miR-451a could induce apoptosis through suppressing BAP31 in CRC. Immunohistochemistry and real-time PCR were used to measure BAP31 expressions in CRC tissues and pericarcinous tissues from 57 CRC patients and CRC cell lines. Dual-luciferase reporter assay was used to detect the binding of miR-451a to BAP31. The expression of BAP31 protein in CRC tissues was significantly higher than that in pericarcinous tissues, which was correlated with distant metastasis and advanced clinical stages of CRC patients. The expression of BAP31 was higher in HCT116, HT29, SW620, and DLD cells than that in the normal colonic epithelial cell line NCM460. The expression of BAP31 was absolutely down-regulated when over-expressing miR-451a in HCT116 and SW620 cells compared with control cells. Mir-451a inhibited the expression of BAP31 by binding to its 5’-UTR. Over-expressing miR-451a or silencing BAP31 suppressed the proliferation and apoptosis of CRC cells by increasing the expressions of endoplasmic reticulum stress (ERS)-associated proteins, including GRP78/BIP, BAX, and PERK/elF2α/ATF4/CHOP, which resulted in increased ERS, cytoplasmic calcium ion flowing, and apoptosis of CRC cells. These changes resulting from over-expressing miR-451a were reversed by over-expressing BAP31 with mutated miR-451a-binding sites. Over-expressing miR-451a or silencing BAP31 inhibited tumor growth by inducing ERS. The present study demonstrated that miR-451a can inhibit proliferation and increase apoptosis through inducing ERS by binding to the 5’-UTR of BAP31 in CRC.
We found several inaccurate descriptions in our recently published paper, A reference human genome dataset of the BGISEQ-500 sequencer [1], thus we would like to make the following corrections. In the Background section, the full name of cPAS should be combinatorial probe-anchor synthesis [2] rather than combined primer anchor synthesis. So the following sentence should be modifed: 1. Sentence.. sequencing library construction and combined primer anchor synthesis (cPAS) for sequencing. should be changed to.. sequencing library construction and combinatorial probe-anchor synthesis (cPAS) for sequencing. In the Background-Sequencing section, the mentioned sequencing technology should be rolling circle amplifcation (RCA) [2] instead of PCR amplifcation. The RCA step is one of Complete GenomicsTM core technologies, which is different from PCR amplifcation. The key advantage of the DNB technology is that every replicated copy is produced from the original genomic fragment, instead of PCR-utilizing sequencing technologies, in which one amplifes from the already amplifed template. Thus two sentences should be modifed accordingly: 2. Sentence.. single-strand circular DNA library was frst PCR-amplifed for 10 minutes.. should be changed to.. single-strand circular DNA library was frst subjected to rolling circle amplifcation (RCA) for 10 minutes.. 3. Sentence After the PCR reaction, 20 μl DNBs stopping buffer was added to terminate the PCR reaction should be changed to After the RCA reaction, 20 μl DNBs stopping buffer was added to terminate the RCA reaction.
Background/Aims: MiRNAs may regulate neurogenic differentiation of adipose-derived stem cells (ADSCs). In this study, we hypothesized that the miR-142-5p can repress the expression of RhoA/ROCK1 pathway on the neurogenesis of ADSCs. Methods: Deregulated miRNA during neurogenic differentiation of ADSCs were identified. The expression of neuron-specific enolase (NSE) and β III tubulin (Neuron-specific class III beta-tubulin) were detected as the markers of neurogenic differentiation by immunostaining and western blot. The targeting of miR-142-5p on RhoA and ROCK1 was verified by dual luciferase assay, qRT-PCR and western blot. The roles of miR-142-5p and the RhoA/ROCK1 signaling pathway were explored by using functional experiments including cell viability and colony formation assays. Results: MiR-142-5p is significantly upregulated during neurogenic differentiation of ADSCs. Knockdown of endogenous miR-142-5p hampered neurogenic differentiation. MiR-142-5p could directly target RhoA and ROCK1 mRNA and repress their expressions, through which it increased the proportion of differentiated cells with positive NSE and β III tubulin. RhoA/ROCK1 signaling pathway is involved in miR-142-5p effect on the process of neurogenic differentiation of ADSCs. Conclusion: Our results demonstrate that miR-142-5p functions as a growth promotive miRNA and plays an important role in neurogenic differentiation by targeting RhoA/ROCK1 in ADSCs.
Background
Resistance to cisplatin results in recurrence or relapse of cervical cancer in women. An understanding of the mechanisms of cisplatin resistance will be important to improve the efficacy of cisplatin treatment. The aim of this study was to investigate the role of microRNA-7-5p (mir-7-5p) in cisplatin-resistant cervical cancer cells in vitro.
Material/Methods
The expression levels of miR-7-5p were detected in cisplatin-resistant cervical cancer cells, HeLa, and SiHa cells (HPV16-positive), and in clinical tissue samples, using miR-7-5p inhibition and a luciferase reporter assay. Fifteen paired cervical cancer tissue samples and adjacent normal cervical tissues were obtained from 15 patients who underwent surgery for cervical cancer. Western blot and flow cytometry were used to investigate cell apoptosis. The expression of mir-7-5p was detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR).
Results
The level of miR-7-5p was increased in cisplatin-resistant HeLa and SiHa cervical cancer cells. Increased expression of miR-7-5p inhibited DNA repair by modulating the expression of poly (ADP-ribose) polymerase 1 (PARP-1), reducing energy consumption, and promoting autophagy via suppression of the expression of Bcl-2. These findings supported that increasing energy generation and reducing energy consumption, resulted in miR-7-5p maintaining energy homeostasis during cisplatin treatment.
Conclusions
The findings of this study showed that there was a protective role of miR-7-5p in cervical cancer cells treated with cisplatin and that miR-7-5p expression maintained energy homeostasis in cisplatin-resistant cervical cancer cells. However, miR-7-5p reduced energy consumption via inhibiting PARP-1 expression, and miR-7-5p increased energy generation by suppressing the expression of Bcl-2.
Staphylococcal enterotoxin A is well known as a superantigen and able to be used for cancer immunotherapy. In this study, recombinant Staphylococcal enterotoxin A was genetically conjugated to epidermal growth factor to produce a chimeric protein recombinant Staphylococcal enterotoxin A–epidermal growth factor expressed in Escherichia coli. The recombinant Staphylococcal enterotoxin A–epidermal growth factor protein was purified using Strep-Tactin affinity chromatography and Endotoxin Removal Resin and identified by sodium dodecyl sulfate-polyacrylamide gel electropheresis and liquid chromatography-tandem mass spectrometry analysis. Furthermore, in vitro experiments showed purified recombinant Staphylococcal enterotoxin A–epidermal growth factor could successfully bind to the human nasopharyngeal carcinoma cell line CNE2, significantly promote the proliferation of human peripheral blood mononuclear cells, and enhance the secretion of several cytokines that have broad antitumor activities, such as interferon-γ, tumor necrosis factor-α, and interleukin-2 . Importantly, recombinant Staphylococcal enterotoxin A–epidermal growth factor significantly inhibited proliferation of CNE2 cells and promoted apoptosis in CNE2 cells when cocultured with peripheral blood mononuclear cells. Finally, both the binding of recombinant Staphylococcal enterotoxin A–epidermal growth factor and the toxicity of recombinant Staphylococcal enterotoxin A–epidermal growth factor-activated peripheral blood mononuclear cells were demonstrated as specific and only effective on high epidermal growth factor receptor-expressing cell lines. In all, our work suggests that recombinant Staphylococcal enterotoxin A–epidermal growth factor serves as a promising novel immunotherapeutic agent. More in vivo and in vitro studies are needed to verify its antitumor potency, as well as investigate the underlying mechanisms in cancer immunotherapy.
SncRNA-Seq has become a routine for sncRNA profiling; however, software packages currently available are either exclusively for miRNA or piRNA annotation (e.g., miRDeep, miRanalyzer, Shortstack, PIANO), or for direct mapping of the sequence reads to the genome (e.g., Bowtie 2, SOAP and BWA), which tend to generate inaccurate counting due to repetitive matches to the genome or sncRNA homologs. Moreover, novel sncRNA variants in the sequencing reads, including those bearing small overhangs or internal insertions, deletions or mutations, are totally excluded from counting by these algorithms, leading to potential quantification bias. To overcome these problems, a comprehensive software package that can annotate all known small RNA species with adjustable tolerance towards small mismatches is needed. AASRA is based on our unique anchor alignment algorithm, which not only avoids repetitive or ambiguous counting, but also distinguishes mature miRNA from precursor miRNA reads. Compared to all existing pipelines for small RNA annotation, AASRA is superior in the following aspects: 1) AASRA can annotate all known sncRNA species simultaneously with the capability of distinguishing mature and precursor miRNAs; 2) AASRA can identify and allow for inclusion of sncRNA variants with small overhangs and/or internal insertions/deletions into the final counts; 3) AASRA is the fastest among all small RNA annotation pipelines tested. AASRA represents an all-in-one sncRNA annotation pipeline, which allows for high-speed, simultaneous annotation of all known sncRNA species with the capability to distinguish mature from precursor miRNAs, and to identify novel sncRNA variants in the sncRNA-Seq sequencing reads.
Availability and Implementation
The AASRA software is freely available at https://github.com/biogramming/AASRA .
A wide variety of clinical conditions, associated with low circulating platelet counts, require platelet transfusion in order to normalize hemostatic function. Although single-donor apheresis platelets bear the lowest risk of transfusion-transmitted infections, pathogen reduction technologies (PRT) are being implemented worldwide to reduce this risk further through inactivation of known, emergent and as yet to be discovered nucleic acid-based pathogens. Human blood platelets are now known to harbor a diverse transcriptome, important to their function and comprised of >5000 protein-coding messenger RNAs and different classes of non-coding RNAs, including microRNAs. Our appreciation of the nucleic acid-dependent functions of platelets is likely to increase. On the other hand, the side effects of PRT on platelet function are underappreciated. Recent evidences suggest that PRT may compromise platelets' responsiveness to agonists, and induce platelet activation. For instance, platelets have the propensity to release proinflammatory microparticles (MPs) upon activation, and the possibility that PRT may enhance the production of platelet MPs in platelet concentrates (PCs) appears likely. With this in mind, it would be timely and appropriate to investigate other means to inactivate pathogens more specifically, or to modify the currently available PRT so to better preserve the platelet function and improve the safety of PCs; platelets' perspective to PRT deserves to be considered.
MicroRNAs (miRNAs) are short ~22-nucleotide noncoding RNA that have been found to influence the expression of many genes and cellular processes by either repressing translation or degrading messenger RNA transcripts. Platelet miRNA expression has been shown to be perturbed during ex vivo storage of platelets and in platelet-associated disorders. Although bioinformatics-based miRNA target predictions have been established, direct experimental validation of the role of miRNAs in platelet biology has been rather slow. Target prediction studies are, nonetheless, valuable in directing the design of appropriate experiments to test specific miRNA:messenger RNA interactions relevant to the underlying mechanisms of platelet function in general and in disease as well as in ex vivo storage-associated "storage lesions," a collective term used to include physiologic, biochemical, and morphologic changes that occur in stored platelets. This brief review will focus on emerging human platelet miRNA studies to emphasize their potential role relevant to transfusion medicine field in terms of regulating platelet signaling pathways, markers of platelet associated disorders, and remote impactors of gene expression (intercellular biomodulators) as well as potential platelet quality markers of storage and pathogen reduction treatments.
Background: MetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors. In this study, we identified neuroblastoma metastamiRs exploiting unique mouse models of favorable and high-risk metastatic human neuroblastoma. Further, we related their deregulation to the modulation of target proteins and established their association with clinical outcomes.
Results: Whole genome miRNA microarray analysis identified 74 metastamiRs across the manifold of metastatic tumors. RT-qPCR on select miRNAs validated profile expression. Results from bio-informatics across the ingenuity pathway, miRCancer, and literature data-mining endorsed the expression of these miRNAs in multiple tumor systems and showed their role in metastasis, identifying them as metastamiRs. Immunoblotting and TMA-IHC analyses revealed alterations in the expression/phosphorylation of metastamiRs’ targets, including ADAMTS-1, AKT1/2/3, ASK1, AURKβ, Birc1, Birc2, Bric5, β-CATENIN, CASP8, CD54, CDK4, CREB, CTGF, CXCR4, CYCLIN-D1, EGFR, ELK1, ESR1, CFOS, FOSB, FRA, GRB10, GSK3β, IL1α, JUND, kRAS, KRTAP1, MCP1, MEGF10, MMP2, MMP3, MMP9, MMP10, MTA2, MYB, cMYC, NF2, NOS3, P21, pP38, PTPN3, CLEAVED PARP, PKC, SDF-1β, SEMA3D, SELE, STAT3, TLR3, TNFα, TNFR1, and VEGF in aggressive cells ex vivo and in a manifold of metastatic tumors in vivo. miRNA mimic (hsa-miR-125b, hsa-miR-27b, hsa-miR-93, hsa-miR-20a) and inhibitor (hsa-miR-1224-3p, hsa-miR-1260) approach for select miRNAs revealed the direct influence of the altered metastamiRs in the regulation of identified protein targets. Clinical outcome association analysis with the validated metastamiRs’ targets corresponded strongly with poor overall and relapse-free survival.
Conclusions: For the first time, these results identified a comprehensive list of neuroblastoma metastamiRs, related their deregulation to altered expression of protein targets, and established their association with poor clinical outcomes. The identified set of distinctive neuroblastoma metastamiRs could serve as potential candidates for diagnostic markers for the switch from favorable to high-risk metastatic disease.
Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen + ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin + UVB light) treatment of platelets
did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.
Lysosomal cathepsins have recently been reported to play crucial roles in the regulation of the mitochondrial death cascade by an unclear mechanism leading to mitochondrial membrane permeabilization. Glycosaminoglycans (GAG) are a family of ionic polysaccharides present at the lysosomal compartment and shown to inhibit lysosomal cathepsin activities. The implication of this family of polysaccharides in the regulation of the pre-mitochondrial death cascade has still not been considered. Here, we demonstrate in a model of skin fibroblasts submitted to oxidative stress that a GAG–mimetic protects the lysosome from membrane disruption, reduces intracellular ROS levels, and inhibits mitochondrial membrane potential collapse, cytochrome c release and caspases-9 and -3 activations without affecting the extrinsic pathway of apoptosis. Heparan sulfate and chondroitin sulfate, but not heparin, showed also protecting effects when assessing key points of the intrinsic pathway of apoptosis. We suggest the existence of molecular links between endogenous GAGs and the regulation of apoptosis.
MicroRNAs are small RNA molecules that modulate protein expression by degrading mRNA or repressing translation. They have been shown to play important roles in hematopoiesis, including embryonic stem cell differentiation, erythropoiesis, granulocytopoiesis/monocytopoiesis, lymphopoiesis, and megakaryocytopoiesis. miR-150 and miR-155 play divergent roles in megakaryocytopoiesis, with the former promoting development of megakaryocytes at the expense of erythrocytes and the latter causing a reduction in megakaryocyte colony formation. Platelets also contain fully functional miRNA machinery, and certain miRNA levels in platelets have been found to coordinate with reactivity to specific agonists and to pathologic states. This review will cover the current state of knowledge of miRNAs in megakaryocytes and platelets and the exciting possibilities for future research.
High-throughput RNA sequencing (RNA-seq) is rapidly emerging as a major quantitative transcriptome profiling platform. Here,
we present DEGseq, an R package to identify differentially expressed genes or isoforms for RNA-seq data from different samples.
In this package, we integrated three existing methods, and introduced two novel methods based on MA-plot to detect and visualize
gene expression difference.
Availability: The R package and a quick-start vignette is available at http://bioinfo.au.tsinghua.edu.cn/software/degseq
Contact: xwwang{at}tsinghua.edu.cn; zhangxg{at}tsinghua.edu.cn
Supplementary information: Supplementary data are available at Bioinformatics online.
Platelets have a crucial role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion, which underlie stroke and acute coronary syndromes. Anucleate platelets contain mRNAs and are capable of protein synthesis, raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs), which are known as key regulators of mRNA translation in other cell types. Further analyses revealed that platelets contain the Dicer and Argonaute 2 (Ago2) complexes, which function in the processing of exogenous miRNA precursors and the control of specific reporter transcripts, respectively. Detection of the receptor P2Y(12) mRNA in Ago2 immunoprecipitates suggests that P2Y(12) expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system.
The hippocampal expression profiles of wild-type mice and mice transgenic for δC-doublecortin-like kinase were compared with
Solexa/Illumina deep sequencing technology and five different microarray platforms. With Illumina's digital gene expression
assay, we obtained ∼2.4 million sequence tags per sample, their abundance spanning four orders of magnitude. Results were
highly reproducible, even across laboratories. With a dedicated Bayesian model, we found differential expression of 3179 transcripts
with an estimated false-discovery rate of 8.5%. This is a much higher figure than found for microarrays. The overlap in differentially
expressed transcripts found with deep sequencing and microarrays was most significant for Affymetrix. The changes in expression
observed by deep sequencing were larger than observed by microarrays or quantitative PCR. Relevant processes such as calmodulin-dependent
protein kinase activity and vesicle transport along microtubules were found affected by deep sequencing but not by microarrays.
While undetectable by microarrays, antisense transcription was found for 51% of all genes and alternative polyadenylation
for 47%. We conclude that deep sequencing provides a major advance in robustness, comparability and richness of expression
profiling data and is expected to boost collaborative, comparative and integrative genomics studies.
Abstract
Introduction: Osteoarthritis (OA) is a widely prevalent joint disease leading to motor disability and pain. Appropriate indicators for identifying patients at risk for this progressive disease, identifying molecular events for detecting early phases of the disease, or biomarkers to screen for treatment responses, however, are lacking. Micro RNAs (miRNAs), which play crucial roles in OA, could be potential biomarkers of OA. Because circulating miRNA levels reflect the disease state, they may be useful for OA screening and as diagnostic tools, reducing the need for invasive procedures and minimizing the cost of current diagnostic methods. Materials and methods: The expression levels of 18 microRNAs (let-7e-5p, miR-21-5p, miR-93-5p, miR-101-3p, miR-103a-3p, miR-130a-3p miR-146a-5p, miR-16-5p, miR-193b-3p miR-199a-3p, miR-210-3p, miR-222-3p, miR-22-3p, miR-27a-3p, miR-27b-3p, miR-335-5p, miR-454-3p, and miR-98-5p) were analyzed by quantitative real-time polymerase chain reaction in the cartilage tissues and serum samples of 28 OA patients and were compared to those of 2 healthy controls. Results: Expression of microRNA-146a-5p was significantly upregulated in the cartilage (p=0.006) and serum (p=0.002) of OA patients. The expression levels of miR-146a-5p in the serum were positively correlated with those in the cartilage (Pearson correlation coefficient R=0.32; p=0.002). Conclusion: miR-146a-5p was significantly overexpressed in patients with OA, both in the articular cartilage tissue and serum, with a positive correlation between the levels in both types of samples. Therefore, the miR-146a-5p serum level could reflect the molecular processes in the cartilage, suggesting its clinical utility as a biomarker for OA management. Implementing noninvasive biomarker using serum miRNAs involves the analysis of the misregulated miRNAs linked to the cartilage pathology.
Immunosuppression is currently a vital pathophysiological characteristic and core problem of sepsis. Apoptosis of T lymphocyte contribute to immunosuppression by decreasing immune effector cells. A report has recently revealed the potential regulatory role of exosomal miRNAs derived from plasma of septic patients on immune system, but the underlying mechanism is unclear. We discovered the antiapoptotic effect of circulating exosomes derived from plasma of septic patients (Sepsis-Exos)on T lymphocytes and further investigated the molecular mechanism. Next-generation sequencing (NGS)indicated that sepsis induces prominent change of exosomal miRNA expression profile, including the overexpressed hsa-miR-7-5p. Gene Bad, which is in the cGMP-PKG signaling pathway, was negatively regulated by hsa-miR-7-5p by dual luciferase reporter assay. Sepsis-Exos were demonstrated to downregulate the mRNA and protein levels of proapoptotic gene Bad, active Caspase-3 and Bax, while upregulate that of antiapoptotic gene Bcl-2 via hsa-miR-7-5p, thus inhibited apoptosis of T lymphocytes induced by lipopolysaccharide (LPS)in vitro. Furthermore, Sepsis-Exos was verified to inhibit T lymphocytes apoptosis during sepsis in vivo, reducing mortality rate of septic model mice. In conclusion, we provide evidence that Sepsis-Exos participate in ameliorating apoptosis of T lymphocytes by directly suppressing Bad via hsa-miR-7-5p.
Aim
Studies have reported that micro (miR)-486-5p plays a crucial part in the progression of leukemia, however, to the best of our knowledge, few studies have been conducted on its mechanism in leukemia. In this study, the mechanism of miR-486-5p in leukemia cells was pointed out and its possible target genes were analyzed for the purpose of providing new therapeutic strategies for treating leukemia patients.
Methods
MiRNA expression of Leukemia cells (K562, Kasumi-1, and THP-1) and primary leukocytes was detected by Real-time Quantitative polymerase chain reaction(qPCR). The activity of the cells was assessed using the Cell Counting Kit-8 (CCK-8). Apoptotic cells were analyzed by a flow cytometer (FCM). Caspase-3 activation in leukemia cells was determined by Western blot. Targetscan 7.2 was used to predict the potential targets of miR-486-5p and further confirmed by dual-luciferase reporter assay.
Result
miR-486-5p was significantly down-regulated in leukemia cells. The over-expression of miR-486-5p notably increased the apoptosis and caspase-3 activity in leukemia cells. There was a predicted interaction site for miR-486-5p in the FOXO1 3′-UTR. Furthermore, this study showed that FOXO1 was significantly up-regulated in leukemia cells, the growth of which was depressed by the up-regulation of miR-486-5p.
Conclusion
miR-486-5p may inhibit the proliferation of leukemia cells and induce apoptosis through targeting FOXO1.
Renal cell carcinoma (RCC) is a common kidney tumor in adults. The role of miR‐486‐5p in RCC is unknown. The aim of our study was to identify new targets regulated by miR‐486‐5p in RCC, to obtain a deeper insight into the network and to better understand the role of these microRNAs and their targets in carcinogenesis of RCC. We performed a series of tests and found consistently lower expression levels of miR‐486‐5p in kidney cancer cells. Restoration of miR‐486‐5p expression in RCC cells could lead to the suppression of cell proliferation and the increase of cell apoptosis. Further studies demonstrated that TGF‐β–activated kinase 1 was a target gene of miR‐486‐5p in kidney cancer cells. It was also shown that C‐C motif chemokine ligand 2 (CCL2) from tumor‐associated macrophages downregulated miR‐486‐5p expression, and miR‐486‐5p inhibited RCC cell proliferation and apoptosis resistance induced by CCL2. The study demonstrates that there are potential diagnosis and therapy values of miR‐486‐5p in RCC. Renal cell carcinoma (RCC) with low levels of miR‐486‐5p expression compared with normal tissues have a relatively poor prognosis, whereas high miR‐486‐5p expression is associated with a long survival rate in patients with RCC. TGF‐β–activated kinase 1 (TAK1) was a target gene of miR‐486‐5p in RCC cells. Low levels of miR‐486‐5p were negatively associated with TAK1 in patients with RCC . miR‐486‐5p could suppress RCC cell proliferation and increase cell apoptosis. C‐C motif chemokine ligand 2 (CCL2) from tumor‐associated macrophages downregulated miR‐486‐5p levels in RCC cells and miR‐486‐5p inhibited cell proliferation and induced apoptosis of RCC cells in the presence of CCL2.
MicroRNAs (miRNAs) play a critical role on biological and cellular processes, the search for functional markers may be of importance for differential diagnosis, prognosis and development of new therapeutic regimens. In this context, we evaluated the bone marrow miRNA profile of Brazilian children exhibiting T or B cell acute lymphoblastic leukemia (T‐ALL or B‐ALL), using massive parallel sequencing, using the HiSeq 2500 platform (Illumina). The differential expression analysis was conducted considering a leave‐one‐out approach and FDR ≤ 0.05. Machine learning algorithms were applied to search for the disease subset biomarkers. Target prediction, functional enrichment and classification of biological categories were also performed. Sixteen miRNAs were differentially expressed between T‐ and B‐ALL, of which 10 (miR‐708‐5p, miR‐497‐5p, miR‐151a‐5p, miR‐151b, miR‐371b‐5p, miR‐455‐5p, miR‐195‐5p, miR‐1266‐5p, miR‐574‐5p, miR‐425‐5p) were downregulated and 6 (miR‐450b‐5p, miR‐450a‐5p, miR‐542‐5p, miR‐424‐5p, miR‐629‐5p, miR‐29c‐5p) were upregulated in childhood T‐ALL. These miRNAs may be used for distinguishing childhood lymphoblastic leukemia subtypes, since it provided the clearly separation of patients in these two distinct groups. Six relevant biological pathways were identified according to their role in leukemia, namely viral carcinogenesis, cell cycle and B cell receptor signaling pathways for induced miRNAs, and TGF‐beta signaling, apoptosis, and NF‐kappa B signaling for the repressed miRNAs, of which several miRNA gene targets participate in cell differentiation and hematopoiesis processes. Machine learning analysis pointed out miR‐29c‐5p expression as the best discriminator between childhood T‐ and B‐ALL, which is involved in calcium signaling, critical for B‐cell lymphocyte fate. Further studies are needed to assure the role of the 16 miRNAs and miR‐29c‐5p on acute lymphoblastic leukemia subtypes and on disease prognosis.
Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) is the most fatal leukemia due to the BCR/ABL fusion protein. This fusion protein could induce the interleukin 6 (IL-6) expression in leukemia stem cells (LSCs), which sustain the stemness by binding IL-6R and activating Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. IL-6R was one of the targets of miR-451a, which was downregulated in LSCs by BCR/ABL. We aimed to investigate the relationship between miR-451a, IL-6R, and BCR/ABL in Ph+ ALL and create a strategy to treat this disease. The expression levels of miR-451a and BCR/ABL of Ph+ ALL patients were examined by real-time quantitative polymerase chain reaction (RT-qPCR); serum IL-6 were tested by enzyme-linked immunosorbent assay. Ph+ ALL cell line SUP-B15 and Ph- ALL cell line Nalm-6 were treated with miR-451a mimic and inhibitor, respectively; the proliferation rate was assessed by CCK-8, the apoptosis rate was tested by Annexin/PI, the expression levels of Bcl-XL, Bax, cyclin D2, and c-myc were examined by qPCR and western blot (WB). The levels of STAT3, p-STAT3, JAK2, and p-JAK2 were tested by WB. We found that BCR/ABL was inversely related with miR-451a and positively related with IL-6 in Ph+ ALL. MiR-451a inhibited the proliferation of SUP-B15 through apoptosis pathway. The oncogene c-myc was downregulated by miR-451a. We confirmed that miR-451a could target IL-6R and inhibit activation of JAK and STAT3. In conclusion, miR-451a is down regulated in Ph+ ALL, and increasing the expression levels of miR-451a in leukemia cells may increase the potential to curing this disease.
Introduction:
Hepatocyte proliferation and apoptosis are critical cellular behaviors in rat liver as a result of a liver injury. Herein, we performed this study in order to evaluate the role of miR-30e and its target Fos-Related Antigen-2 (FOSL2) in septic rats through the JAK/STAT signaling pathway.
Methods:
Rat models of sepsis were induced by cecal ligation and puncture. Enzyme-linked immunosorbent assay (ELISA) was performed to access serum levels of lipopolysaccharide (LPS), inflammatory factors, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to confirm the successful establishment of the model. The hepatocytes were subject to miR-30e mimics, miR-30e inhibitors or siRNA-FOSL2. The expressions of miR-30e, FOSL2, apoptosis- and, JAK/STAT signaling pathway-related genes in liver tissues and hepatocytes were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. MTT assay and flow cytometry were performed to evaluate hepatocyte viability and apoptosis, respectively.
Results:
The results obtained revealed that in the septic rats, serum levels of inflammatory factors, LPS, ALT and AST, as well as the expression of FOSL2 were elevated and the JAK/STAT signaling pathway was activated, while there was a reduction in the expression of miR-30e. An initial bioinformatics prediction followed by a confirmatory dual-luciferase reporter assay determined that miR-30e targeted and negatively regulated FOSL2 expression. MiR-30e inhibited the activation of JSK2/STAT3 signaling pathway by reducing FOSL2 expression, while miR-30e enhanced hepatocyte proliferation and decreased hepatocyte cell apoptosis in septic rats.
Conclusion:
These findings indicated that miR-30e may serve as an independent therapeutic target for sepsis, due to its ability to inhibit apoptosis and induce proliferation of hepatocytes by targeted inhibition of FOSL2 through the JAK/STAT signaling pathway.
Tuberculosis continues to be one of the deadliest infectious disease worldwide. MicroRNAs (miRNAs) are small non-coding entities that play critical role as post-transcriptional regulators and are transcriptionally deregulated upon mycobacterial infection. In this study, we found significant upregulation of hsa-let-7b-5p in Mycobacterium tuberculosis (MTB) infected THP-1 human macrophages. Concomitantly, we detected the reduced level of Fas protein, one of the targets of hsa-let-7b-5p, in MTB infected THP-1 macrophages. Using luciferase assay a direct interaction between hsa-let-7b-5p and the Fas 3'-untranslated region (UTR) was established. Inhibition of hsa-let-7b-5p augmented the apoptosis of THP-1 cells enabling enhanced clearance of MTB. Our findings suggest that hsa-let-7b-5p helps intracellular survival of MTB in THP-1 cells by down-regulating Fas protein level. This highlights hsa-let-7b-5p as a potential therapeutic target for tuberculosis treatment.
Hydroquinone (HQ), one of the major metabolic products of benzene, is a carcinogen, which induces apoptosis and inhibit proliferation in lymphoma cells. microRNA-7-5p (miR-7-5p), a tumor suppressor, participates in various biological processes including cell proliferation and apoptosis regulation by repressing expression of specific oncogenic target genes. To explore whether miR-7-5p is involved in HQ-induced cell proliferation and apoptosis, we assessed the effect of miR-7-5p overexpression on induction of apoptosis analyzed by FACSCalibur flow cytometer in transfection of TK6 cells with miR-7-5p mimic (TK6- miR-7-5p). We observed an increased apoptosis by 25.43% and decreased proliferation by 28.30% in TK6-miR-7-5p cells compared to those negative control cells (TK6-shNC) in response to HQ treatment. Furthermore, HQ might active the apoptotic pathway via partly downregulation the expression of BRCA1 and PARP-1, followed by p53 activation, in TK6-miR-7-5p cells. In contrast, attenuated p53 and BRCA1 expression was observed in shPARP-1 cells than in NC cells after HQ treatment. Therefore, we conclude that HQ may activate apoptotic signals via inhibiting the tumor suppressive effects of miR-7-5p, which may be mediated partly by upregulating the expression of PARP-1 and BRCA1 in control cells. The increase of miR-7-5p expression further intensified downregulation of PARP-1 and BRCA1 in TK6-miR-7-5p cells, resulting in an increase of apoptosis and proliferation inhibited.
MicroRNAs (miRNAs) are essential for the maintenance of podocyte homeostasis. Emerging evidence demonstrated a protective role of miRNA-30a, a member of miR-30 family in podocyte injury. However, the role of other members of miR-30 family in podocyte injury is unclear. The present study was undertaken to investigate the contribution of miR-30e in the pathogenesis of podocyte injury induced by aldosterone (Aldo), as well as the underlying mechanism. Following Aldo treatment, miR-30e was dose-and time-dependently reduced. Notably, overexpression of miR-30e remarkably attenuated Aldo-induced apoptosis in podocytes. In agreement with this finding, miR-30e silencing led to significant podocyte apoptosis. Mitochondrial dysfunction (MtD) has been shown as an early event in Aldo-induced podocyte injury. Here we found that overexpressing miR-30e improved Aldo-induced MtD while miR-30e silencing resulted in MtD. Next, we found that miR-30e could directly target BCL2/adenovirus E1B interacting protein 3-like (BNIP3L) gene. Aldo remarkably enhanced BNIP3L expression in podocytes and silencing BNIP3L largely abolished Aldo-induced MtD and cell apoptosis. On the contrary, overexpression of BNIP3L induced MtD and apoptosis in podocytes. Taken together, these findings demonstrated that miR-30e protected mitochondria and podocytes against Aldo challenge by targeting BNIP3L.
Platelet storage lesions seriously affect the quality of stored platelets, even causing them to be ineffective in vivo after transfusion. Past researches have been focused on what mechanism(s) cause the formation of storage lesions. One proposed mechanism is microRNAs (miRNAs)-based molecular regulation of the platelet mRNAs that are relevant to the storage lesion. Platelets continue to translate proteins from mRNA while in a storage environment. A strong correlation exists between the platelet transcriptome and its subsequent proteomic profile, which supports de novo platelet translational capabilities. Thus, miRNA may play a crucial role in platelet biology during storage conditions. Importantly, this suggests the exciting possibility of post-transcriptional regulation of gene expression in platelets that are in storage. Given this, the differential profiling of miRNAs could be a useful tool in identifying changes to ex vivo stored platelets. Any identified miRNAs could then be considered as potential markers to assess the viability of platelet concentrates. The present review summarizes the current experimental and clinical evidence that clarifies the role miRNAs play during platelet ex vivo storage.
The transfusion of platelet concentrates (PCs) is mainly used for treatment of thrombocytopenic, trauma or surgery patients. The integrity and safety of these platelet preparations, however, is compromised by the presence of pathogens, such as viruses, bacteria and parasites. The transfer of allogeneic donor leukocytes contaminating PCs can also potentially cause adverse reactions in recipients. These considerations prompted the development and implementation of pathogen reduction technologies (PRT), which are based on chemically induced cross-linking and inactivation of nucleic acids. While the incumbent PRT may provide some protection against transfusion-transmitted infections, they are ineffective against infectious prions and may not inactivate other emerging pathogens. In addition, the safety of PRT concerning platelet viability and function has been questioned in several reports. Recent studies suggest that PRT, such as Intercept, may adversely affect the messenger RNA (mRNA) and microRNA content of platelets, as well as their functional integrity, which may compromise the clinical benefits of PRT. Here, we will discuss about the peculiarities of studying the effects of PRT on platelets, which will need to be taken into account in future studies aimed to characterize further, and polish, the rugged side of this otherwise useful and potentially important approach in transfusion medicine. This article is protected by copyright. All rights reserved.
There are many sources of systematic variation in cDNA microarray experiments which affect the measured gene expression levels
(e.g. differences in labeling efficiency between the two fluorescent dyes). The term normalization refers to the process of
removing such variation. A constant adjustment is often used to force the distribution of the intensity log ratios to have
a median of zero for each slide. However, such global normalization approaches are not adequate in situations where dye biases
can depend on spot overall intensity and/or spatial location within the array. This article proposes normalization methods
that are based on robust local regression and account for intensity and spatial dependence in dye biases for different types
of cDNA microarray experiments. The selection of appropriate controls for normalization is discussed and a novel set of controls
(microarray sample pool, MSP) is introduced to aid in intensity-dependent normalization. Lastly, to allow for comparisons
of expression levels across slides, a robust method based on maximum likelihood estimation is proposed to adjust for scale
differences among slides.
Recent reports from Europe have advocated the use of bacterial culturing of platelets on Day 2 or 3 of storage to extend the shelf life of platelets to 7 days, thereby reducing the outdating of platelets and preserving a limited medical resource. To assess the optimal timing, the necessary sensitivity, and the possible efficacy of bacterial detection, the bacterial growth characteristics were reviewed in 165 platelet units, each inoculated on the day of collection with one of the following organisms: Bacillus cereus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens, Staphylococcus aureus, and Staphylococcus epidermidis from four previously published studies.
Quantitative culture data from inoculated platelet concentrates from five sites and four studies were combined into one database and analyzed for bacterial concentration thresholds (> or =10(1), > or =10(2), > or =10(3), > or =10(4), > or =10(5) CFU/mL) by day of storage.
All examples of B. cereus, P. aeruginosa, K. pneumoniae, S. marcescens, and S. aureus had concentrations > or =10(2) CFU per mL by Day 3 after inoculation. By Day 4, all units with these organisms contained > or =10(5) CFU per mL. Units contaminated with S. epidermidis showed slower and more varied growth. By Day 3 after inoculation, 81.3 percent had 10(2) CFU per mL. By Day 4 after inoculation, 46 (95.8%) of 48 units had concentrations > or =10(2) CFU per mL.
These experiments suggest that an assay capable of detecting 10(2) CFU per mL on Day 3 of storage would detect the vast majority of bacterially contaminated platelet units, prevent many cases of platelet-associated bacterial sepsis, and provide a scientific basis for the extension of the current platelet storage time. It would be expected that a rare, slow-growing organism could escape such a detection scheme.
Platelets are anucleate blood cells that play a crucial role in thrombosis and hemostasis. Despite their lack of nuclear DNA, platelets contain significant amounts of microRNA (miRNA) that may have vital functions in post-transcriptional gene regulation. Here, we combined comprehensive miRNA expression profiling by quantitative PCR with target prediction analysis for the most abundant miRNAs in human platelets. A network composed of predicted platelet miRNA target genes was then constructed, using annotations available in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. In addition, we evaluated possible differences in miRNA levels between resting and thrombin-stimulated platelets. We identified 281 transcripts, including 228 mature miRNAs and 53 minor miRNAs (or miR*), of which six miRNAs (miR-15 a, miR-339-3 p, miR-365, miR-495, miR-98, and miR-361-3 p) were up- or down-regulated in activated human platelets (P ≤ 0.001). A redundancy-reduced network was established that encompassed 246 genes in five statistically significant functional clusters representing platelet miRNA regulating pathways. Comparison of the 246 network genes with the platelet mRNA expression data available at ArrayExpress database confirmed that most of these genes (89%) are expressed in human platelets. In conclusion, this work affirms a recent microarray study reporting a wide-spread existence of miRNAs in human platelets. Further, we observed that thrombin stimulation was associated with altered levels of some miRNAs in platelets. The proposed functional network, combining computational prediction analysis with annotations from experimental observations, may in addition provide some information about probable miRNA target pathways in human platelets.
MicroRNAs (miRNAs) regulate cell physiology by altering protein expression, but the biology of platelet miRNAs is largely unexplored. We tested whether platelet miRNA levels were associated with platelet reactivity by genome-wide profiling using platelet RNA from 19 healthy subjects. We found that human platelets express 284 miRNAs. Unsupervised hierarchical clustering of miRNA profiles resulted in 2 groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed (DE) between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. Using whole genome mRNA expression data on these same subjects, we computationally generated a high-priority list of miRNA-mRNA pairs in which the DE platelet miRNAs had binding sites in 3'-untranslated regions of DE mRNAs, and the levels were negatively correlated. Three miRNA-mRNA pairs (miR-200b:PRKAR2B, miR-495:KLHL5, and miR-107:CLOCK) were selected from this list, and all 3 miRNAs knocked down protein expression from the target mRNA. Reduced activation from platelets lacking PRKAR2B supported these findings. In summary, (1) platelet miRNAs are able to repress expression of platelet proteins, (2) miRNA profiles are associated with and may predict platelet reactivity, and (3) bioinformatic approaches can successfully identify functional miRNAs in platelets.
As part of its risk management process, Canadian Blood Services (CBS) constructed mathematical models of how newly emerging pathogens might affect blood transfusion recipients.
CBS convened an expert panel including medical, health economics, analytical, risk management, and insurance professionals to examine multiple data sources. The model for emerging pathogen risk included separate modules to calculate the frequency and severity of infections from transfusion-transmitted agents that could cause either acute transient or chronic persistent infection. Important model input variables were annual number of components transfused, the presumed incidence and prevalence of a new agent, the time interval of recipient risk, recipient age and sex, projected recipient survival, rate of secondary infection, pathogen-induced morbidity, and the associated medical costs of such morbidity.
In the 5-year time frame considered in the model, it was estimated that approximately 3500 recipient infections (two-SD range of 0 to 11,370 infections) could occur from an emerging pathogen that establishes a chronic infection in donors, with 60% of these due to red blood cell transfusion. The medical costs associated with recipient outcomes due to a catastrophic emerging pathogen could be lowered by 20% if an effective pathogen reduction method for either platelets or plasma were in place.
This modeling exercise offers a framework for other blood services to construct similar models. It also provides a useful way to model the implementation of new blood safety interventions (e.g., pathogen reduction) on emerging pathogen risk.
Transfusion of platelet concentrates (PCs) is the basic treatment for severe platelet disorders. PCs carry the risk of pathogen transmission, especially bacteria. Pathogen reduction (PR) by addition of photochemical reagents and irradiation with visible or ultraviolet (UV) light can significantly reduce this risk. We present a novel approach for PR in PCs employing UVC light alone.
UVC PR was evaluated by bacteria and virus infectivity assays. PC quality was investigated by measuring pH, lactate, glucose, hypotonic shock response, platelet aggregation, CD62P expression, and annexin V binding as in vitro parameters. The impact of UVC PR on the platelet proteome was assessed by differential in-gel electrophoresis and compared with changes caused by UVB and gamma-irradiation, respectively.
Vigorous agitation of loosely placed PCs generated thin fluid layers that allow penetration of UVC light for inactivation of the six bacteria and six of the seven virus species tested. HIV-1 was only moderately inactivated. UVC light at the dose used (0.4 J/cm(2)) had a minor impact on in vitro parameters and on storage stability of treated PCs. Proteome analysis revealed a common set of 92 (out of 793) protein spots being affected by all three types of irradiation. Specific alterations were most pronounced for gamma-irradiation (45 spots), followed by UVB (11 spots) and UVC (2 spots).
UVC irradiation is a potential new method for pathogen reduction in PCs. The data obtained until now justify further development of this process.
Enucleated platelets (PLTs) utilize posttranscriptional gene (mRNA) regulation (PTGR) for their normal morphologic and physiologic functions, which are altered in their ex vivo storage, also collectively referred to as storage lesions. While cellular micro-RNAs (miRNAs) play a significant role in posttranscriptional gene (mRNA) regulation by binding to their target mRNAs, comprehensive analysis of apoptosis-associated miRNAs and global changes in their profiles during PLT storage have not been evaluated to date.
In this report room temperature-stored PLTs of Days 0, 2, and 9 were analyzed by differential profiling for 52 apoptosis-associated human miRNAs. After total RNA extraction from the samples, a membrane array-based miRNA analysis was carried out. Prediction of target genes was performed by bioinformatics-based approaches.
Our analysis revealed that during storage, Let-7a, -7c, -7e, -7f, -7g, and -7i miRNA profiles of the PLTs were barely detectable, while levels of miR-150, -151, -152, -184, -188, -196a, -197, and -202 remained at high levels in PLTs. The rest of the miRNA levels were in between. However, two miRNAs, Let-7b and miR-16, distinctly demonstrated an increasing trend while miR-7 and miR-145 showed a decreasing profile during PLT storage. For these four miRNAs, we also identified their potential target mRNAs.
Overall, these results confirm the fact that miRNAs do exist in PLTs, and among 52 apoptosis-specific miRNAs studied, only a few selected miRNAs did perturb during PLT storage. Future experimental evaluation of these miRNA-target mRNA interactions will provide new insights into the molecular mechanisms of PLT storage-associated lesions.
To examine if different pathogen-reduction technologies (PRTs) induce different degrees of platelet (PLT) storage lesion.
Twenty-seven split triple-dose apheresis PLTs were PRT treated using ultraviolet light with either riboflavin (M) or psoralen (I) or remained untreated (C). Samples taken on days (d) 0 to 8 were analysed for PLT count, blood gas (pH, pO(2) and pCO(2)), metabolism (lactate, glucose, ATP content), in vitro function [swirling, hypotonic shock response (HSR) and aggregation], activation (p-selectin expression) and cellular integrity (JC-1 signal, annexin A5 release).
Platelet counts of all study groups remained unchanged during storage indicating that PRT treatment did not induce relevant cell lysis. Although M units demonstrated the highest values for HSR until d5, PRT treatment lowered all parameters examined with significant differences to untreated controls by d7 of storage. During final storage, M was significantly superior over I for HSR, aggregation with TRAP-6 as agonist (collagen was similar), annexin A5 release and JC-1 signal. Regarding blood gas and metabolic analysis, the most evident effect of PRT was an elevated glycolytic flux combined with higher acidity due to increased lactate accumulation. Most likely due to impaired O(2) consumption, pH and ATP decreased more rapidly in I relative to C and M.
Pathogen reduction technology-treated PLTs remained comparable to untreated units throughout 7 days of storage. Mitochondria-based oxidative respiration appeared up-regulated after the riboflavin-based PRT. Compared to the psoralen-based PRT, this resulted in significantly better ATP maintenance and in vitro function during the last storage period (d7, d8).
The relationship between the platelet storage lesion (PSL) and programmed cell death (apoptosis) is poorly understood. Nevertheless, there is some experimental evidence that platelets contain most of the components of the apoptosis machinery and both the apoptotic process and the PSL lead to platelet activation and microvesiculation with expression of phosphatidyl serine (PS) on the outer layer of cell membrane, a hallmark of all nucleated cells. The PS exposure is believed to contribute to the development of inflammatory or immunomodulation process, to the regulation of haemostatic balance and the ultimate clearance of dead or fragmented cells from the circulation. While there is no doubt that apoptosis, as a form of genetically encoded programmed cell death in nucleated cells, is triggered by several signalling stimuli at the nuclear level, there is some doubt as to whether platelets, as enucleated cells have retained the memory of the "parental" megakaryocytes for apoptosis or whether platelet mitochondrial DNA has a major role in both the apoptotic process and the PSL. The storage lesion occurs during processing and storage subsequent to mechanical trauma, hypoxic conditions or exposure to cold. In this brief report some observational evidence is provided in support of the notion that the PSL and apoptosis may be related to each other, despite the fact that, in contrast to the 'parental' megakarocyte, the platelets appear to survive upon stimulation with a high concentration of protein kinase inhibitors such as staurosporine (STS), in the presence of cycloheximide (CHX) which inhibit protein synthesis. This is a model which is often used to regulate the level of survival signals. The possible relevance of platelet microvesiculation to transfusion practice is briefly discussed.
There are many sources of systematic variation in cDNA microarray experiments which affect the measured gene expression levels (e.g. differences in labeling efficiency between the two fluorescent dyes). The term normalization refers to the process of removing such variation. A constant adjustment is often used to force the distribution of the intensity log ratios to have a median of zero for each slide. However, such global normalization approaches are not adequate in situations where dye biases can depend on spot overall intensity and/or spatial location within the array. This article proposes normalization methods that are based on robust local regression and account for intensity and spatial dependence in dye biases for different types of cDNA microarray experiments. The selection of appropriate controls for normalization is discussed and a novel set of controls (microarray sample pool, MSP) is introduced to aid in intensity-dependent normalization. Lastly, to allow for comparisons of expression levels across slides, a robust method based on maximum likelihood estimation is proposed to adjust for scale differences among slides.