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MiR-1468 promotes cell proliferation, cell cycle progression, colony formation and inhibits apoptosis in HCC cells in vitro. a Hep3B and MHCC-97 L cells that were transfected with corresponding miRNA vectors were subjected to qRT-PCR for miR-1468 expression. Overexpression of miR-1468 promoted cell proliferation (b, c), colony formation (d), cell cycle progression (e) and inhibited apoptosis (f) in Hep3B cells, while down-regulation of miR-1468 inhibited cell proliferation (b, c), colony formation (d), cell cycle progression (e) and promoted apoptosis (f) in MHCC-97 L cells. g Western blot analysis of cycle regulator Cyclin D1 and p21, apoptosis-related protein Bcl2/Bax expression in the presence and absence of miR-1468. n = six independent experiments. *P < 0.05, **P < 0.01
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Background:
Accumulating evidence confirm that aberrant microRNAs (miRNAs) expression contributes to hepatocellular carcinoma (HCC) development and progression. Previous study reported that miR-1468 showed an up-regulated tendency and might be a potential prognostic biomarker in HCC samples derived from TCGA database. However, the role of miR-1468...
Contexts in source publication
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... studies confirm that the target genes of miR- 1468 are significantly enriched in PPAR signaling path- way and CITED2 is involved in PPAR-γ effects on HCC growth [16,29]. Therefore, we explored the PPAR-γ/ AKT signaling by western blot. As shown in Fig. 7a, overexpression of miR-1468 significantly inhibited, while miR-1468 knockdown promoted PPAR-γ/AKT pathway in HCC cells (P < 0.05, Fig. 7a), with change of p-AKT level rather than total AKT (P < 0.05, Fig. 7a). Moreover, CITED2 or UPF1 overexpression promoted PPAR-γ/ AKT pathway in HCC cells (Additional file 2: Figure S2). To investigate whether PPAR-γ mediated miR-1468- induced biological function in HCC cells, we treated miR-1468-overexpressing Hep3B cells with the agonist rosiglitazone (40 μM). We found that rosiglitazone at least partially inhibited miR-1468-induced cell prolifera- tion, cell cycle progression, colony formation and apop- tosis resistant in HCC cells (P < 0.05, Fig. 7b-f ). Conversely, T0070907 (50 μM), an antagonist of PPAR- γ, rescued the effects of miR-1468 knockdown on biological function of HCC (P < 0.05, Fig. 7b-f ) in miR- 1468-suppressive MHCC-97 L cells. Moreover, alterna- tion of PPAR-γ also abolished the effects of miR-1468 on cell cycle and apoptosis-related proteins (P < 0.05, Fig. 7g). In conclusion, our results indicate that PPAR-γ/ (See figure on previous page.) Fig. 4 CITED2 and UPF1 are direct targets of miR-1468 in HCC cells. a miR-1468 and its putative binding sequences in the 3'-UTR of CITED2 and UPF1. The mutant binding site was generated in the complementary site for the seed region of miR-1468. b miR-1468 overexpression significantly suppressed, while miR-1468 loss increased the luciferase activity that carried wild-type (wt) but not mutant (mt) 3'-UTR of CITED2 or UPF1. c Hep3B and MHCC-97 L cells that were transfected with precursor miR-1468 and miR-1468 inhibitors (anti-miR-1468), respectively, were subjected to qRT-PCR for CITED2 and UPF1 mRNA expression. d miR-1468 overexpression reduced the expression of CITED2 and UPF1 protein in Hep3B cells and miR-1468 knockdown increased the level of CITED2 and UPF1 protein in MHCC-97 L cells. e, f Immunofluorescence staining of CITED2 and UPF1 after transduction of miR-1468. n.s, no significance, *P < ...
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... further investigate the biological function of miR- 1468 in HCC, miR-1468 was stably overexpressed in Hep3B cells by lentivirus system and knocked down in MHCC-97 L cells, which contained different endogen- ous miR-1468 levels. As measured by qRT-PCR, we confirmed that miR-1468 was effectively upregulated in Hep3B or downregulated in MHCC-97 L cells (P < 0.05, Fig. 2a). CCK8 and EdU proliferation assays found that miR-1468 overexpression enhanced cell proliferation (P < 0.05, Fig. 2b, c). The colony formation assay revealed that ectopic expression of miR-1468 signifi- cantly increased cell colonies (P < 0.05, Fig. 2d). As determined by flow cytometry, overexpression of miR- 1468 promoted cell cycle transition from G1 to S phase (P < 0.05, Fig. 2e) and apoptosis resistance (P < 0.05, Fig. 2f). In addition, immunoblotting analysis confirmed that up-regulated miR-1468 level obviously increased Cyclin D1 and Bcl-2 expression, while reduced the levels of p21 and Bax (P < 0.05, respectively, Fig. 2g). In accordance, miR-1468 knockdown inhibited the growth and induced apoptosis of MHCC-97 L cells (P < 0.05, Fig. 2b-g). Thus, these data demonstrate that miR-1468 regulates HCC cell proliferation, cell cycle progression, colony formation and apoptosis in ...
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... further investigate the biological function of miR- 1468 in HCC, miR-1468 was stably overexpressed in Hep3B cells by lentivirus system and knocked down in MHCC-97 L cells, which contained different endogen- ous miR-1468 levels. As measured by qRT-PCR, we confirmed that miR-1468 was effectively upregulated in Hep3B or downregulated in MHCC-97 L cells (P < 0.05, Fig. 2a). CCK8 and EdU proliferation assays found that miR-1468 overexpression enhanced cell proliferation (P < 0.05, Fig. 2b, c). The colony formation assay revealed that ectopic expression of miR-1468 signifi- cantly increased cell colonies (P < 0.05, Fig. 2d). As determined by flow cytometry, overexpression of miR- 1468 promoted cell cycle transition from G1 to S phase (P < 0.05, Fig. 2e) and apoptosis resistance (P < 0.05, Fig. 2f). In addition, immunoblotting analysis confirmed that up-regulated miR-1468 level obviously increased Cyclin D1 and Bcl-2 expression, while reduced the levels of p21 and Bax (P < 0.05, respectively, Fig. 2g). In accordance, miR-1468 knockdown inhibited the growth and induced apoptosis of MHCC-97 L cells (P < 0.05, Fig. 2b-g). Thus, these data demonstrate that miR-1468 regulates HCC cell proliferation, cell cycle progression, colony formation and apoptosis in ...
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... further investigate the biological function of miR- 1468 in HCC, miR-1468 was stably overexpressed in Hep3B cells by lentivirus system and knocked down in MHCC-97 L cells, which contained different endogen- ous miR-1468 levels. As measured by qRT-PCR, we confirmed that miR-1468 was effectively upregulated in Hep3B or downregulated in MHCC-97 L cells (P < 0.05, Fig. 2a). CCK8 and EdU proliferation assays found that miR-1468 overexpression enhanced cell proliferation (P < 0.05, Fig. 2b, c). The colony formation assay revealed that ectopic expression of miR-1468 signifi- cantly increased cell colonies (P < 0.05, Fig. 2d). As determined by flow cytometry, overexpression of miR- 1468 promoted cell cycle transition from G1 to S phase (P < 0.05, Fig. 2e) and apoptosis resistance (P < 0.05, Fig. 2f). In addition, immunoblotting analysis confirmed that up-regulated miR-1468 level obviously increased Cyclin D1 and Bcl-2 expression, while reduced the levels of p21 and Bax (P < 0.05, respectively, Fig. 2g). In accordance, miR-1468 knockdown inhibited the growth and induced apoptosis of MHCC-97 L cells (P < 0.05, Fig. 2b-g). Thus, these data demonstrate that miR-1468 regulates HCC cell proliferation, cell cycle progression, colony formation and apoptosis in ...
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... further investigate the biological function of miR- 1468 in HCC, miR-1468 was stably overexpressed in Hep3B cells by lentivirus system and knocked down in MHCC-97 L cells, which contained different endogen- ous miR-1468 levels. As measured by qRT-PCR, we confirmed that miR-1468 was effectively upregulated in Hep3B or downregulated in MHCC-97 L cells (P < 0.05, Fig. 2a). CCK8 and EdU proliferation assays found that miR-1468 overexpression enhanced cell proliferation (P < 0.05, Fig. 2b, c). The colony formation assay revealed that ectopic expression of miR-1468 signifi- cantly increased cell colonies (P < 0.05, Fig. 2d). As determined by flow cytometry, overexpression of miR- 1468 promoted cell cycle transition from G1 to S phase (P < 0.05, Fig. 2e) and apoptosis resistance (P < 0.05, Fig. 2f). In addition, immunoblotting analysis confirmed that up-regulated miR-1468 level obviously increased Cyclin D1 and Bcl-2 expression, while reduced the levels of p21 and Bax (P < 0.05, respectively, Fig. 2g). In accordance, miR-1468 knockdown inhibited the growth and induced apoptosis of MHCC-97 L cells (P < 0.05, Fig. 2b-g). Thus, these data demonstrate that miR-1468 regulates HCC cell proliferation, cell cycle progression, colony formation and apoptosis in ...
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... further investigate the biological function of miR- 1468 in HCC, miR-1468 was stably overexpressed in Hep3B cells by lentivirus system and knocked down in MHCC-97 L cells, which contained different endogen- ous miR-1468 levels. As measured by qRT-PCR, we confirmed that miR-1468 was effectively upregulated in Hep3B or downregulated in MHCC-97 L cells (P < 0.05, Fig. 2a). CCK8 and EdU proliferation assays found that miR-1468 overexpression enhanced cell proliferation (P < 0.05, Fig. 2b, c). The colony formation assay revealed that ectopic expression of miR-1468 signifi- cantly increased cell colonies (P < 0.05, Fig. 2d). As determined by flow cytometry, overexpression of miR- 1468 promoted cell cycle transition from G1 to S phase (P < 0.05, Fig. 2e) and apoptosis resistance (P < 0.05, Fig. 2f). In addition, immunoblotting analysis confirmed that up-regulated miR-1468 level obviously increased Cyclin D1 and Bcl-2 expression, while reduced the levels of p21 and Bax (P < 0.05, respectively, Fig. 2g). In accordance, miR-1468 knockdown inhibited the growth and induced apoptosis of MHCC-97 L cells (P < 0.05, Fig. 2b-g). Thus, these data demonstrate that miR-1468 regulates HCC cell proliferation, cell cycle progression, colony formation and apoptosis in ...
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... further investigate the biological function of miR- 1468 in HCC, miR-1468 was stably overexpressed in Hep3B cells by lentivirus system and knocked down in MHCC-97 L cells, which contained different endogen- ous miR-1468 levels. As measured by qRT-PCR, we confirmed that miR-1468 was effectively upregulated in Hep3B or downregulated in MHCC-97 L cells (P < 0.05, Fig. 2a). CCK8 and EdU proliferation assays found that miR-1468 overexpression enhanced cell proliferation (P < 0.05, Fig. 2b, c). The colony formation assay revealed that ectopic expression of miR-1468 signifi- cantly increased cell colonies (P < 0.05, Fig. 2d). As determined by flow cytometry, overexpression of miR- 1468 promoted cell cycle transition from G1 to S phase (P < 0.05, Fig. 2e) and apoptosis resistance (P < 0.05, Fig. 2f). In addition, immunoblotting analysis confirmed that up-regulated miR-1468 level obviously increased Cyclin D1 and Bcl-2 expression, while reduced the levels of p21 and Bax (P < 0.05, respectively, Fig. 2g). In accordance, miR-1468 knockdown inhibited the growth and induced apoptosis of MHCC-97 L cells (P < 0.05, Fig. 2b-g). Thus, these data demonstrate that miR-1468 regulates HCC cell proliferation, cell cycle progression, colony formation and apoptosis in ...
Context 8
... further investigate the biological function of miR- 1468 in HCC, miR-1468 was stably overexpressed in Hep3B cells by lentivirus system and knocked down in MHCC-97 L cells, which contained different endogen- ous miR-1468 levels. As measured by qRT-PCR, we confirmed that miR-1468 was effectively upregulated in Hep3B or downregulated in MHCC-97 L cells (P < 0.05, Fig. 2a). CCK8 and EdU proliferation assays found that miR-1468 overexpression enhanced cell proliferation (P < 0.05, Fig. 2b, c). The colony formation assay revealed that ectopic expression of miR-1468 signifi- cantly increased cell colonies (P < 0.05, Fig. 2d). As determined by flow cytometry, overexpression of miR- 1468 promoted cell cycle transition from G1 to S phase (P < 0.05, Fig. 2e) and apoptosis resistance (P < 0.05, Fig. 2f). In addition, immunoblotting analysis confirmed that up-regulated miR-1468 level obviously increased Cyclin D1 and Bcl-2 expression, while reduced the levels of p21 and Bax (P < 0.05, respectively, Fig. 2g). In accordance, miR-1468 knockdown inhibited the growth and induced apoptosis of MHCC-97 L cells (P < 0.05, Fig. 2b-g). Thus, these data demonstrate that miR-1468 regulates HCC cell proliferation, cell cycle progression, colony formation and apoptosis in ...
Citations
... (Rao et al., 2020) 2020). Besides miR-660-5p, other microRNAs, including miR-126-5p and miR-1468, are also frequently upregulated in hepatocellular carcinoma and promote tumor cell proliferation by activating AKT signaling (Ai et al., 2022;Liu, 2018). Up-frameshift protein 1 (UPF1) and carboxyterminal domain 2 (CITED2) are the direct downstream targets of miR-1468, and they mediate peroxisome proliferator-activated receptor-γ (PPAR-γ)/AKT signaling activation (Liu, 2018). ...
... Besides miR-660-5p, other microRNAs, including miR-126-5p and miR-1468, are also frequently upregulated in hepatocellular carcinoma and promote tumor cell proliferation by activating AKT signaling (Ai et al., 2022;Liu, 2018). Up-frameshift protein 1 (UPF1) and carboxyterminal domain 2 (CITED2) are the direct downstream targets of miR-1468, and they mediate peroxisome proliferator-activated receptor-γ (PPAR-γ)/AKT signaling activation (Liu, 2018). Similarly, miRNA-126-5p positively regulates TDO2 (tryptophan 2,3-dioxygenase), a critical metabolic enzyme in the tryptophan-kynurenine pathway that regulates cell growth and proliferation of HCCLM3 and HepG2 cells. ...
... Molecular pathway level analysis found that the dysregulated expression of DEGs in ICC was associated with the metabolism of aberrant tRNA, amino acid, and lipoprotein 29 . The increasing pieces of evidence also revealed that aberrantly expressed miRNAs were common in various types of human cancer and play important roles in tumorigenesis [30][31][32][33] . Thank to the high-throughput sequencing of small RNAs, 39 differentially expressed miRNAs were filtrated from the ICC tissues. ...
Intrahepatic cholangiocarcinoma (ICC) is the second common primary hepatic malignancy tumor. In this study, an integrative analysis of differentially expressed genes (DEGs) and miRNAs from the ICC onset and adjacent normal tissues were performed to explore the regulatory roles of miRNA–mRNA interaction. A total of 1018 DEGs and 39 miRNAs were likely involved in ICC pathogenesis, suggesting the changes in cell metabolism in ICC development. The built network indicated that 30 DEGs were regulated by 16 differentially expressed miRNA. The screened DEGs and miRNA together were probably considered the biomarkers of ICC, and their important roles in ICC pathogenesis remain to be elucidated. This study could provide a good basis to uncover the regulatory mechanism of miRNA and mRNAs in ICC pathogenesis.
... Some miRNAs are differentially expressed in serum samples of HBV related HCC, CHB patients, and healthy individuals have been reported [9,10]. Additionally, accumulating evidence indicates that miRNAs can act as oncogenic, or tumor suppressive factors involved in HCC progress [11,12]. ...
... The transition from epithelial to mesenchymal cells is promoted through TGF-β induced SMAD2/SMAD3 activation. The activated proteins in turn operate as transcription factors promoting the transcription of genes involved in the dissolution of cell junctions and detachment from adjacent tissue [130]. Qu et al., also demonstrated intercellular communication of HCC cells by exosomes promoting their mutual invasion and migration. ...
Hepatocellular carcinoma (HCC) is the most frequent liver cancer with high lethality and low five-year survival rates leading to a substantial worldwide burden for healthcare systems. HCC initiation and progression are favored by different etiological risk factors including hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, non-/and alcoholic fatty liver disease (N/AFLD), and tobacco smoking. In molecular pathogenesis, endogenous alteration in genetics (TP53, TERT, CTNNB1, etc.), epigenetics (DNA-methylation, miRNA, lncRNA, etc.), and dysregulation of key signaling pathways (Wnt/β-catenin, JAK/STAT, etc.) strongly contribute to the development of HCC. The multitude and complexity of different pathomechanisms also reflect the difficulties in tailored medical therapy of HCC. Treatment options for HCC are strictly dependent on tumor staging and liver function, which are structured by the updated Barcelona Clinic Liver Cancer classification system. Surgical resection, local ablative techniques, and liver transplantation are valid and curative therapeutic options for early tumor stages. For multifocal and metastatic diseases, systemic therapy is recommended. While Sorafenib had been the standalone HCC first-line therapy for decades, recent developments had led to the approval of new treatment options as first-line as well as second-line treatment. Anti-PD-L1 directed combination therapies either with anti-VEGF directed agents or with anti-CTLA-4 active substances have been implemented as the new treatment standard in the first-line setting. However, data from clinical trials indicate different responses on specific therapeutic regimens depending on the underlying pathogenesis of hepatocellular cancer. Therefore, histopathological examinations have been re-emphasized by current international clinical guidelines in addition to the standardized radiological diagnosis using contrast-enhanced cross-sectional imaging. In this review, we emphasize the current knowledge on molecular pathogenesis of hepatocellular carcinoma. On this occasion, the treatment sequences for early and advanced tumor stages according to the recently updated Barcelona Clinic Liver Cancer classification system and the current algorithm of systemic therapy (first-, second-, and third-line treatment) are summarized. Furthermore, we discuss novel precautional and pre-therapeutic approaches including therapeutic vaccination, adoptive cell transfer, locoregional therapy enhancement, and non-coding RNA-based therapy as promising treatment options. These novel treatments may prolong overall survival rates in regard with quality of life and liver function as mainstay of HCC therapy.
... Peroxisome proliferator-activated receptors a (PPARa) and c (PPARc) played an vital role in inhibiting cholesterol biosynthesis [51]. Previous studies have shown that PPARa and PPARc are involved in HCC progression [52,53]. Thus, PPARa and PPARc are considered as potential drug targets for cancer patients [54]. ...
Hepatocellular carcinoma (HCC) represents the most important type of liver cancer, the 5-year survival rate for advanced HCC is 2%. The heterogeneity of HCC makes previous models fail to achieve satisfactory results. The role of Cholesterol-based metabolic reprogramming in cancer has attracted more and more attention. In this study, we screened cholesterol metabolism-related genes (CMRGs) based on a systematical analysis from TCGA and GEO database. Then, we constructed a prognostic signature based on the screened 5 CMRGs: FDPS, FABP5, ANXA2, ACADL and HMGCS2. The clinical value of the five CMRGs was validated by TCGA database and HPA database. HCC patients were assigned to the high-risk and low-risk groups on the basis of median risk score calculated by the five CMRGs. We evaluated the signature in TCGA database and validated in ICGC database. The results revealed that the prognostic signature had good prognostic performance, even among different clinicopathological subgroups. The function analysis linked CMRGs with KEGG pathway, such as cell adhesion molecules, drug metabolism-cytochrome P450 and other related pathways. In addition, patients in the high-risk group exhibited characteristics of high TP53 mutation, high immune checkpoints expression and high immune cell infiltration. Furthermore, based on the prognostic signature, we identified 25 most significant small molecule drugs as potential drugs for HCC patients. Finally, a nomogram combined risk score and TNM stage was constructed. These results indicated our prognostic signature has an excellent prediction performance. This study is expected to provide a potential diagnostic and therapeutic strategies for HCC.
... Notably, because blood samples can be collected noninvasively and frequently, and because miRNAs are tissue-specific, circulating miRNAs in serum or exosomes may be more relevant indicators than those in tissues. Several miRNA Prognosis miRNA-92a ↑ HBV-associated HCC and others [281] miRNA-487a ↑ HBV-associated HCC and others [282] miRNA-1468 ↑ HBV-associated HCC and others [283] miRNA-221 ↑ HBV-associated HCC and others [284] miRNA-137 ↓ HBV-associated HCC and others [285] [290] profiles have recently been found as HCC biomarkers ( Table 5). The potential value of circulating miRNA-122 and let-7 in early-stage HCC detection has been revealed, with serum levels in HBV patients with precancerous nodules and early HCC having comparable sensitivity to that of AFP [269]. ...
... Several miRNAs have shown promise as prognostic indicators of HCC prognosis. Recent research has linked high expression of miRNA-92a [281], miRNA-487a [282], miRNA-1468 [283] or miRNA-221 [284] ( Table 5). Downregulation of miRNA-137 [285], miRNA-194 [286], miRNA-940 [287], or miRNA-33a [288], on the other hand, resulted in a poor prognosis in HCC. ...
Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide. HCC initiation, progression, and therapy failure are all influenced by various variables, including microRNAs (miRNAs). miRNAs are short non-coding RNA sequences that modulate target mRNA expression by deteriorating or repressing translation. miRNAs play an imperative role in HCC pathogenesis by triggering the induction of cancer stem cells (CSCs) and their proliferation, while also delaying apoptosis, sustaining the cell cycle, and inspiring angiogenesis, invasion, and metastasis. Additionally, miRNAs modulate crucial HCC-related molecular pathways such as the p53 pathway, the Wnt/β-catenin pathway, VEGFR2, and PTEN/PI3K/AKT pathway. Consequently, the goal of this review was to give an up-to-date overview of oncogenic and tumor suppressor (TS) miRNAs, as well as their potential significance in HCC pathogenesis and treatment responses, highlighting their underpinning molecular pathways in HCC initiation and progression. Similarly, the biological importance and clinical application of miRNAs in HCC are summarized.
... [10] Various miRNAs, including miR-32a-5p, miR-122, miR-221, and miR-1468, have potential as prognostic biomarkers for HCC, and upregulation of these miRNAs indicates poor prognosis in HCC patients. [11][12][13][14] miR-20a has significantly higher expression in ovarian cancer, lung cancer, colorectal cancer, cervical cancer, gastric cancer, and head and neck cancer according to The Cancer Genome Atlas (TCGA) database (https://cancer genome.nih.gov/) statistics, and it exhibits potential for clinical application as a novel diagnostic biomarker and therapeutic target. ...
Background:
MicroRNA-20a (miR-20a) is dysregulated in many types of malignancies, including human hepatocellular carcinoma (HCC), but its expression level and functional significance in HCC are still disputed. We aimed to study the role of miR-20a-5p in HCC and its downstream molecular mechanisms.
Methods:
We used real-time polymerase chain reaction to detect the expression of miR-20a-5p and runt-related transcription factor 3 (RUNX3) in HCC and paraneoplastic tissue and in transfected Huh7 and Highly metastatic human hepatocellular carcinoma cells (MHCC97H) cells. A live cell workstation was used to observe the proliferation and migration of transfected cells. The invasiveness of transfected cells was verified by Transwell assay. Cell apoptosis was detected by flow cytometry. The expression levels of proteins after transfection were measured using simple western immunoblot measurements. Gene expression profiles between HCC and normal samples were obtained from The Cancer Genome Atlas. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were processed by the database for annotation, visualization and integrated discovery. Potential target genes of miR-20a-5p were predicted to further investigate how miR-20a-5p regulates epithelial-mesenchymal transition (EMT) in HCC.
Results:
MiR-20a-5p was significantly highly expressed in HCC tissues, and overexpression of miR-20a-5p significantly promoted HCC cell proliferation, migration, and invasion and inhibited apoptosis in vitro. The protein expression of E-cadherin was decreased and that of vimentin was increased after overexpression of miR-20a-5p in HCC cells. After the intersection of of miRDB, miRTarBase, and TargetScan, we obtained 397 target genes and finally focused on RUNX3. RUNX3 was not only reduced in HCC specimens but also drastically reduced in HCC cells overexpressing miR-20a-5p. RUNX3 expression decreased with elevated miR-20a-5p, which activated downstream EMT signaling and further promoted cell proliferation, migration, and invasion.
Conclusions:
Since RUNX3 is involved in EMT in HCC, as proven by previous research, our findings provide further evidence for a novel regulatory pathway comprising the miR-20a/RUNX3/EMT axis that upregulates EMT signaling and enhances the migration of HCC cells.
... Although miRNAs are non-coding RNAs, by binding with the 3'-untranslated region (3'-UTR) of target mRNAs, miRNAs promote mRNA cleavage or block mRNA translation, thereby controlling the expression of certain target proteins in cells (14,15). In addition, miRNAs also serve important roles during the occurrence, development and metastasis of tumors (16,17). Numerous studies have reported the tumor-suppressive role of miR-193a-5p. ...
Epithelial ovarian cancer (EOC), a gynecological tumor, is associated with high mortality. MicroRNAs (miRs) serve a crucial role in EOC; however, the mechanisms underlying the effect of miRNA‑193a‑5p in EOC are not completely understood. Therefore, the present study aimed to investigate the expression levels of miR‑193a‑5p in serum samples of patients with EOC and to determine the role of miR‑193a‑5p in EOC. Reverse transcription‑quantitative PCR was used to analyze the expression levels of miR‑193a‑5p in serum samples of patients with EOC and EOC cell lines. The effects of miR‑193a‑5p and RB binding protein 6, ubiquitin ligase (RBBP6) on the biological functions of EOC were determined by conducting a series of in vitro cell function experiments. The results indicated that the expression levels of miR‑193a‑5p were significantly decreased in serum samples obtained from patients with EOC and EOC cell lines compared with healthy individuals and normal cells, respectively. Further investigations indicated that RBBP6 was a target gene of miR‑193a‑5p. The expression levels of RBBP6 were significantly increased in patients with EOC compared with healthy individuals. In addition, in vitro analysis suggested that miR‑193a‑5p mimic significantly decreased SKOV3 cell proliferation, migration and invasion, and promoted SKOV3 cell apoptosis compared with the control and mimic‑negative control groups. In addition, RBBP6 overexpression reversed miR‑193a‑5p mimic‑mediated effects. In conclusion, the results of the present study suggested that downregulated expression levels of miR‑193a‑5p may serve an inhibitory role in EOC by inhibiting cell proliferation and metastasis, and promoting apoptosis.
... Specifically, miRNAs are involved in the regulation of lipid metabolism-related proteins, thus playing vital roles in the development and progression of HCC [9][10][11]. The transcription and expression of miRNAs are regulated by a variety of inflammatory cytokines [12]. ...
Background. Abnormal lipid metabolism is closely associated with the invasiveness and metastasis of cancer. Fatty acid-binding proteins (FABPs) play essential roles in lipid metabolism, and miRNAs can affect lipid metabolism by targeting FABPs. However, the exact mechanism is unknown. Methods. FABP1 expression in HCC tissues was analyzed by immunochemistry with tissue microarrays. The lipid content was detected by Oil Red O staining, and the interaction between FABP1 and free fatty acid (FFA) was studied by a labeling and tracking method. miRNA arrays were used to detect the expression of miRNAs in IL-6-stimulated HCC cells. miR-603 expression was verified by qPCR. The proteins were checked by Western blot analysis. Gain and loss function evaluation was assessed by lentivirus and miRNA mimic transfection in Huh-7 cells, while reactive oxygen species (ROS) were detected by fluorescence. Results. FABP1 expression was significantly decreased in approximately 90% (81/90) of HCC patients. FABP1 expression in adjacent tissues was closely associated with overall survival. Meanwhile, lipid was abundant in the adjacent tissues, yet significantly reduced in HCC tissues. FABP1 and FFA can promote each other for being uptaken by Huh-7 cells. FABP1 overexpression induced apoptosis and inhibited the proliferation, migration, invasion, and metastasis of Huh-7 cells. IL-6 treatment affected the expression of miRNAs, and miR-603 was overexpressed in HCC tissues. Also, miR-603 overexpression promoted the proliferation, migration, invasion, and metastasis of Huh-7 cells. Bioinformatic analysis predicted that miR-603 targets the 3-UTR region of FABP1. However, miR-603 overexpression inhibited the expression of the FABP1 but increased the CPT1A, PPAR-α, and SREBP1 expressions. FABP1 overexpression reduced ROS in HCC cells, while miR-603 can reverse these effects. Conclusion. Our results indicate that in the pathogenesis of HCC, IL-6 induces miR-603 expression, which subsequently inhibits FABP1 expression, promotes the lipid metabolism- and synthesis-related proteins, and finally increases the cellular oxidative stress level and leads to the metastasis of HCC.
1. Introduction
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide [1]. The invasiveness and metastatic potential are two key factors affecting the prognosis of patients with HCC, yet the mechanism remains largely unknown [2]. In terms of dietary structure, lipid metabolism homeostasis plays a central role in preventing HCC, yet its breakdown is a key risk factor associated with the development and progression of HCC [3]. The synthesis, transport, and catabolism of fatty acids are regulated by fatty acid-binding proteins (FABPs), which function as sensors to regulate the homeostasis of lipid metabolism. There are 12 members of the FABP family, which are primarily involved in peripheral free fatty acid (FFA) uptake, transport, fatty acid synthesis, lipid processing, storage, oxidative decomposition, and output.
The liver plays an essential role in fatty acid metabolism, and abnormal lipid metabolism is closely associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and HCC [4, 5]. FABP1 is a special FABP primarily found in the liver, which has two fatty acid-binding sites and a strong affinity for fatty acids. FABP1 is overexpressed in the cytoplasm of HCC cells [6]. microRNA (miRNA) is an endogenous noncoding RNA that participates in the negative regulation of target genes by binding to the 3-UTR (i.e., untranslated region) of mRNA. Studies have shown that miRNAs play important roles in the regulation of lipid metabolism [7, 8].
Specifically, miRNAs are involved in the regulation of lipid metabolism-related proteins, thus playing vital roles in the development and progression of HCC [9–11]. The transcription and expression of miRNAs are regulated by a variety of inflammatory cytokines [12]. IL-6 is an important cytokine in the pathogenesis of HCC [13]. However, it is unknown whether IL-6 can regulate FABP1 and related miRNAs during the development of HCC. In the current study, we systematically investigated the role of the IL-6/miR-603 signaling in regulating the pathogenesis of HCC by acting on FABP1, thereby providing new perspectives on the pathogenesis and treatment of HCC.
2. Materials and Methods
2.1. HCC Tissue Specimens
A total of 18 pairs of HCC and adjacent healthy tissues were obtained from patients of Wuxi People’s Hospital (Wuxi, China), who were diagnosed with HCC and received surgery between July 2016 and September 2018. The patients have not received any chemotherapy before the surgery. The experiment was approved by the Ethics Committee of Nanjing Medical University. Informed consent was obtained from all patients.
2.2. Tissue Array
The tissue array chip was purchased from Shanghai Zuocheng Biotechnology Co., Ltd. (China). The chip contained 90 cases of HCC tissues with corresponding adjacent tissues.
2.3. Immunochemistry
Tissue specimens were sliced at 4-5 μm. Antigen retrieval was performed with a citrate buffer (pH 6.0) at 90°C. The slides were blocked with goat serum (Beyotime Biotechnology, Shanghai, China) before the primary FABP1 antibody was added at a ratio of 1 : 100 (Sigma-Aldrich, St. Louis, MO, USA). After incubating overnight at 4°C, the slides were incubated with goat anti-rabbit secondary antibody (CWBio, Beijing, China) and developed with 3,3-diaminobenzidine (DAB) (Beyotime Biotechnology, Shanghai, China). Hematoxylin (Servicebio, Wuhan, China) was used for nuclear counterstaining.
To evaluate the stained tissues, ten fields of view were randomly selected under 400x magnification, and 200 cells were randomly picked from each field. The staining intensity () was defined as no stain (one point), mild positive staining (light yellow, two points), moderate positive staining (brown or dark yellow, three points), or strong positive staining (tan or dark brown, four points). The percentage of positive cells () was evaluated based on the following: was 0 points, was one point, was two points, was three points, and was four points. The . All histopathological sections were read, diagnosed, and recorded by two senior pathologists with more than 3 years of combined experience.
2.4. Oil Red O Staining of Liver Tissues
The solution was made by mixing 6 ml Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) stock solution with 4 ml distilled water. After mixing for 10 min, the staining buffer was added dropwise onto the tissue for a 5-10 min incubation period. Excess staining buffer was removed with a solution of 60% Oil Red O and isopropanol. The tissues were washed with distilled water and counterstained with hematoxylin (Servicebio, Wuhan, China).
2.5. Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was extracted from tissues according to the TRIzol protocol (Invitrogen, Carlsbad, CA, USA). First, 1 μg of total RNA was used to generate cDNA according to the PrimeScript RT kit (TaKaRa, Shiga, Japan). Next, 2 μl of cDNA was used in the real-time qPCR system. U6 was used as the internal reference, and the relative expression of miR-603 was calculated with the method. Each experiment was repeated in triplicate.
2.6. Cell Culture, Induction, and Microarray Analysis
The human hepatocyte cell lines, Huh-7 and HepG2, were purchased from Cell Bank of Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai China). Cells were cultured in high-sugar DMEM medium (Gibco, Gaithersburg, MD, USA) with 10% fetal bovine serum (FBS, Biological Industries, Cromwell, CT, USA) with 100 U/ml streptomycin and 100 U/ml penicillin (HyClone Company, Logan, UT, USA) in a 37°C humidified incubator with 5% CO2.
For IL-6 treatment experiments, the Huh-7 cells were treated with 10 ng/ml IL-6 (Sigma-Aldrich, St. Louis, MO, USA) for 72 h. After the incubation, total RNA or protein was collected for miRNA array or Western blot detections.
The total RNA of Huh-7 cell was extracted, and the miRNA microarray analysis was performed by Guangzhou Ribobio Co., Ltd. (Guangdong Province, China). One microgram of total RNA was added into a nuclease-free RNA sample PCR tube, and the small RNAs (<300 nucleotides) were separated using the MilliporeSigma Centriplus Centrifugal Concentrators Microcon YM-100 (MilliporeSigma, Burlington, MA) and adding poly(A) to the 3 end of the small RNA for hybridization at 37°C. After hybridization, dyes labeled with specific flash biotin were used, followed by scanning and analyzing the data by Affymetrix Gene Chip™ Command Console Software (AGCC).
2.7. Transfection
Huh-7 or HepG2 cells were seeded in six-well plates to reach about 70% confluency. For overexpression experiments, 50 nM miR-603 mimics (Ribobio, Guangzhou, China) and 5 μl Lipofectamine® RNAiMAX (Invitrogen, Carlsbad, CA, USA) were added into the cells for 6 h in Opti-MEM medium (Gibco, Gaithersburg, MD, USA). A nonspecific mimic was used as a negative control (NSM). After 6 h of incubation, the medium was replaced with a complete medium for 72 h for subsequent experiments.
2.8. Lentiviral Infection
Huh-7 cells were seeded into six-well plates and allowed to reach 30% confluency. Approximately 60 μl of HiTransG A (Genechem Co., Ltd., Shanghai, China), 4 μl TU/ml LV-FABP1 lentivirus (Genechem Co., Ltd., Shanghai, China), and 4 μl TU/ml FABP1 RNAi lentivirus (Genechem Co., Ltd., Shanghai, China.) were added into the 1.5 ml complete medium; the scramble sequence was used as a negative control (NC). After 12 h of incubation, the medium was replaced with complete medium for 72 h for subsequent experiments.
2.9. FFA Treatment and Oil Red O Staining of Cells
FFA was prepared with 1 : 2 ratio of palmitic acid (PA, Sigma-Aldrich, St. Louis, MO, USA) and oleic acid (OA, Sigma-Aldrich, St. Louis, MO, USA). The FFA was dissolved in 0.1% diethylpyrocarbonate- (DEPC-) treated water (HyClone Company, Logan, UT, USA) containing 0.1 mM NaOH and 1% BSA (Absin, Shanghai, China). OA and PA were stored at 20 mM and 10 mM at -20°C and dissolved in a 75°C water bath before use.
After treating the cells in 12-well plates, the cell supernatants were removed. Cells were washed three times with PBS (HyClone Company, Logan, UT, USA) and fixed with 4% neutral formaldehyde for 10 min. Next, the fixed cells were thoroughly washed with PBS and 60% isopropanol for 10 s. Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) was added for 10 min and then washed with 60% isopropanol for 5 s. Finally, the cells were stained with hematoxylin (Servicebio, Wuhan, China) for 10 min, and the optical intensity was measured using a spectrophotometer at 500 nm (Thermo Fisher Scientific).
2.10. FABP1 Labelization and Colocalization
For enhanced green fluorescent protein- (EGFP-) labeled FABP1 protein induction, the cells were cultured in 24-well plates and treated with 175 μg/ml EGFP-labeled FABP1 protein (FABP1-EGFP) (Public Protein/Plasmid Library, Nanjing, China), and the EGFP protein was used as control; 24 h later, the cells were treated with Golgi apparatus or lysosome tracker as followed.
Golgi apparatus tracker red (Beyotime Biotechnology, Shanghai, China) work fluid was prepared according to the manufacturer’s protocol. Cells were washed with Hank’s balanced salt solution with Ca2+ and Mg2+ (Beyotime Biotechnology, Shanghai, China) and incubated with Golgi-Tracker red work fluid at 4°C for 30 min, and then, the cells were washed with DMEM three times and incubated with DMED at 37°C for 30 min. Cells were observed under SP8 laser scanning confocal microscopy (Leica, Gaman), and the fluorescence intensity was measured by ImageJ software.
Lysosome tracker red (Beyotime Biotechnology, Shanghai, China) work fluid was prepared according to the manufacturer’s protocol. Cells were incubated with lysosome tracker red work fluid at 37°C for 30 min; then, the cells were washed with DMEM for three times and incubated with DMED at 37°C for 30 min. Cells were observed under SP8 laser scanning confocal microscopy (Leica, Gaman), and the fluorescence intensity was measured by ImageJ software.
2.11. Western Blot Analysis
Huh-7 or HepG2 cells were collected in radioimmunoprecipitation assay (RIPA) (Beyotime Biotechnology, Shanghai, China) lysis buffer containing 1% phenylmethylsulphonyl fluoride (PMSF) (Beyotime Biotechnology, Shanghai, China) on ice for 10 min. The cell lysates were removed and placed into 1.5 ml Eppendorf tubes and centrifuged at 12,000 rpm for 15 min. Next, the supernatants were used to determine the total protein concentration using the bicinchoninic acid (BCA) (Beyotime Biotechnology, Shanghai, China) assay. Protein samples (25 μg) were loaded into each well and separated with 5% concentrated gels and 12% separation gels. After blocking the membranes with 5% defatted milk, rabbit anti-human FABP1 (1 : 1000, SAB1410361, Sigma-Aldrich), mouse anti-human caspase 3 (1 : 1000, MAB10753 Sigma-Aldrich), mouse anti-human CPT1A (1 : 1000, ab128568, Abcam), rabbit anti-human PPAR-α (phospho S12, 1 : 1000, ab3484, Abcam), and rabbit anti-human SREBP1 (1 : 1000, ab191857, Abcam) antibodies were added and incubated at 4°C overnight. Next, the membranes were incubated with goat anti-rabbit (12348, Sigma-Aldrich) or goat anti-mouse (12349, Sigma-Aldrich) secondary antibody (1 : 5000) for 2 h at room temperature and developed with enhanced chemiluminescence (ECL) kit (Millipore, Burlington, MA, USA). The gels were imaged using the Syngene gel imager (Frederick, MD, USA).
2.12. Cell Proliferation
Huh-7 cells transfected with miR-603 mimics or the FABP1 overexpression lentivirus, along with the negative control group, were seeded in 96-well plates with 10⁴ cells/well, with three replicates in each group. After 24 h in complete medium, cell proliferation was detected with the Lights’ EdU Apollo567 kit (RiboBio, Guangzhou, China), according to the manufacturer’s protocol. The .
2.13. Wound Healing Migration
Huh-7 cells transfected with miR-603 mimics or FABP1 overexpression lentivirus, along with the negative control groups, were seeded into six-well plates to a confluency of 80-90%. All experiment was performed in triplicate. First, a scratch was created by a 200 μl tip at the bottom of the six-well plates. Next, a serum-free medium was added into the wells. Photos were taken every 12 h, beginning at 0 h for the control.
2.14. Reactive Oxygen Species (ROS) Detection
Cells were treated with FFA for 24 h in six-well plates. Next, 33 μM dichlorodihydrofluorescein diacetate (DCFH-DA, Jiancheng Bioengineering Institute, Nanjing, China) was added into cells for 1 h. After the cells were collected by trypsinization and centrifugation at 5 min for 1000 rpm, the samples were read in a fluorescence microplate reader. Each sample was measured three times.
2.15. Statistical Analysis
Data were presented as the (SD). Comparisons between counted data were performed using the homogeneity test of variance and independent sample -tests or nonparametric tests. Kaplan-Meier plots were used for survival analysis. values < 0.05 were considered statistically significant. The analyses were performed using SPSS 21.0 software (IBM, Chicago, IL, USA).
3. Results
3.1. FABP1 and Fatty Acid Expression in HCC and Adjacent Tissues
Immunohistochemistry revealed that FABP1 expression is significantly lower in HCC tissues, as compared with adjacent tissues in nearly 90% (81/90) of cases (Figures 1(a) and 1(b)). HCC was further classified using the TNM stage according to the postoperative pathological features. Independent of the TNM stage, FABP1 expression was always lower than that of adjacent tissues (Figure 1(c)). Next, the correlation between FABP1 expression and overall survival was investigated. In the adjacent tissues, FABP1 expression was significantly correlated with overall patient survival (), as shown in Figure 1(d). Next, Oil Red O staining was used to check the lipid content in HCC and adjacent liver tissues. The adjacent tissues had an abundance of lipid, while lipid content in HCC tissues was low (Figure 1(e)).
(a)
... Currently, this molecule is known to play a critical role in cell proliferation and differentiation by promoting a proliferative, undifferentiated cell state [22]. Further, accumulating studies have been reported that UPF1 is dysregulated and plays important role in various cancers, including hepatocellular cancer [23][24][25][26][27], colorectal cancer [28][29][30][31], gastric cancer [32], lung adenocarcinoma [33,34], pancreatic cancer [35], inflammatory myofibroblastic tumor [36], thyroid cancer [37], ovarian cancer [38], glioma [39] and prostate cancer [40] (Table 1). ...
... Accumulating reports revealed that lncRNAs are involved in various regulations of many cancer processes [53,54]. Recently, researches have demonstrated that UPF1 could exert oncogenic or tumor-suppressive function in various cancers through interacting with lncRNA and MicroRNA, including UCA1 [23], miR-1468 [24], SNHG6 [26][27][28], NR4A1AS [30], MALAT1 [32], ZFPM2-AS1 [34], DLX6-AS1 [37], DANCR [38] and PVT1 [39]. Better understanding of the mechanistic model of UPF1-lncRNA in human cancers may shed new light on tumor pathogenesis and molecular treatment (Fig. 1). ...
... One of the key regulatory mechanisms linked to HCC is the miR-1468/UPF1, CITED2/PPAR-γ/AKT network revealed by Liu et al. [24]. CITED2 and UPF1 were identified as direct downstream targets of miR-1468 in HCC cells, and were inversely correlates with miR-1468. ...
Recently, Up-frameshift protein 1 (UPF1) is reported to be downregulated in various cancers and its low expression is closely correlated with poor prognosis. UPF1 is well known as a master regulator of nonsense-mediated mRNA decay (NMD), which serves as a highly conserved mRNA surveillance process protecting cells from aberrant toxic transcripts. Due to dysfunction of UPF1, NMD fails to proceed, which contributes to tumor initiation and progression. This review shows a brief summary of the aberrant expression, functional roles and molecular mechanisms of UPF1 during tumorigenesis. Increasing evidence has indicated that UPF1 could serve as a potential biomarker for cancer diagnosis and treatment for future clinical applications in cancer.
