| MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcrip-tionally regulate the expression of many target genes via mRNA degradation or translation inhibition. These RNAs have been shown to participate in various cellular and physiological processes;, including cellular development, apoptosis, proliferation, and differentiation. Hepatocellular carcinoma (HCC) is the sixth most common cancer. Chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) resulting in chronic liver diseases are the major causative factors for HCC. But the current therapeutic options for HCC patients are limited, there is an urgent need to analyze the molecular oncogenic mechanisms of HCC to determine novel targets for specific systemic therapy and to detect novel biomarkers for early diagnosis. Many studies have shown that miRNAs are involved in the modulation of gene expression and replication of HBVand play a crucial role in host-virus interactions. The deregulated miRNAs also participate in HCC initiation and progression by functioning as oncogenes or tumor suppressor genes by targeting various genes involved in cancer-related signaling pathways. The role of deregulated miRNA in host-virus interactions and HCC development suggested that miRNAs may serve as therapeutic targets or as tools.In the first section, this research screened an constructed hsa-miRNA expression library in the cell transient transfection model to identify miRNAs with the potential of interfering with HBV replication, and chose the representative miRNAs for molecular mechanisms research of anti-HBV. We used HepG2.2.15 as low HBV replication cell model to screen the miRNAs from the hsa-miRNA expression library constructed by ourselves and identified five miRNAs related to HBV replication. HBsAg level detection of the cell culture after transfection indicated that let7g, miR-101, miR-345 have some promotive effects on HBsAg expression, whereas miR-34a, miR-92b have the opposite effect. We then transfected the five miRNA expression vector and HBV expression vector into HepG2 cell select HBV replication-related miRNA by detecting HBsAg/HBeAg expression. The results showed that miR-34a, but not other miRNAs. could inhibit HBsAg expression. So we selected miR-34a as a candidate to study the role and the mechanisms of miR-34a in HBV replication. We analyzed effect of miR-34a on HBV replication and expression by detecting the HBsAg, HBV DNA and the intracellular HBV RNA, HBV replicative intermediates, HBcAg. The results showed that overexpressed miR-34a level inhibited HBV replication and expression, while decreased miR34a level promoted HBV replication and expression in a persistent HBV expression cell line HepG2.2.15.We further investigated the molecular mechanism of miR-34a in the regulation of HBV replication and expression. After excluding the possibility of miR-34a directly targeting HBV transcripts, we supposed that miR-34a may regulate HBV replication and expression by influencing HBV promoters or enhancers through targeting HBV transcription-related factors. The host target gene of miR-34a were predicted using bioinformatics software and the results contain several genes participated transcriptional regulation or important signal pathway, including HNF1 A, FOXA1, HNF4A, HNF4G as the essential cell regulatory factor in HBV replication. The interactions between these miR-34a and its target genes were verified using luciferase reporter gene assay.The result indicated that miR-34a has interaction with HNF4A 3’-UTR, HNF4G 3’-UTR. To further study the effect of miR-34a on HNF4A mRNA and HNF4G mRNA protein in cells, we found that miR-34a overexpression inhibit the expression of HNF4A mRNA and protein. We also study the effect of miR-34a on HBV promoters or enhancers. We cloned all HBVpromoters and enhancers into luciferase reporter pGL3-Basic and transfected them with miR-34a mimics into cells, the dual-luciferase reporter assay results showed that miR-34a could inhibit the activity of HBV Enhancer Ⅱ, PreS1 promoter, PreS2/S promoter. These above results demonstrated that HNF4A is the mediator of miR-34a inhibiting HBV replication and expression.In the second section, to clarify the biologic function of miR-34a in HCC. Its mimics and inhibitors was transfected into HepG2 and Bel7404 cells. Subsequently HCC cell proliferation and migration were examined by MTT assay, colony formation assay and wound healing assay, respectively. These data show that enhanced miR-34a expression in cultured HepG2 and Bel7404 cells by transfection with miR-34a mimics decreased tumor cell proliferation, migration, we found that the level of miR-34a and cell migration and invasion in several human cell lines were positively correlated. In order to identify the target genes and explore the molecular mechanisms of miR-34a. After analyzed target genes of miR-34a with the application of bioinformatics tools, We found SOX4 could be a potential target gene of miR-34a. Real-time PCR and western blot results showed that HepG2 cells transfected with miR-34a mimics have reduced expression of target genes SOX4. Further investigation revealed that miR-34a significantly repressed the activity of luciferase carrying the 3’UTR of SOX4.We identified SOX, a known tumor oncogene that has been reported to suppress ovarian cance cells proliferation and migration, as a direct target of miR-34a. We showed that SOX4 inhibited cell migration and invasion of HCC cells. Sox4 was able to abrogate the effect of miR-34a on cell migration and invasion partially. In addition, a microarray analysis revealed that miR-34a could regulate many migration and invasion related genes. In addition, we further demosteated that decreased miR34a level promoted β-catenin expression level,and overexpresion of SOX4 also lead to the higher P-catenin expression level. It showed that down-regulation of miR-34a might promote SOX4 expression and activated the Wnt/β-catenin pathway, finally, led to cell proliferation. |
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