Hbv Replication Mirna Expression Spectrum Analysis And Study Of Biological Function Of Mir - 192

Hepatitis B virus﹙HBV﹚ infection is one of mainly risk factors for hepato- cellular carcinoma﹙HCC﹚. It is increasingly clear that microRNAs﹙miRNAs﹚ play a vital role in regulating replication and expression of HBV. Therefore, it is significant to study the function of miRNAs in the progression of HBV-related HCC﹙HBV-HCC﹚ for uncovering the pathogenic mechanism of HBV. In this study, we profiled the aberrant expression of miRNAs in HBV cell models by microarray technology and predicted the targets by bioinformatics softwares. The expression of the miRNAs correlated to replication of HBV was profiled, and live-specific miR-192 whose expression was directly correlated to replication of HBV was subsequently revealed. Our study systematically elucidated the molecular mechanism by which HBV modulated the expression of miR-192 and its inhibition to the expression of RB1.1. Screen and validation of miRNAs related to replication of HBVTo obtain the expression profile of miRNAs in HBV cell models, HBV high replication cell model﹙HepAD38 cells﹚, HBV low replication cell model﹙2.2.15 cells﹚ and control cells﹙HepG2 cells﹚ were used for determining miRNAs aberrantly expressed in HBV cell models by microarray technology. Based on the identification of replication and expression of HBV in these cells, the total RNAs extracted from these cells were hybridized with miRNA microarrays, and analyzed the expression profile of miRNAs. The results demonstrated that the qualities of the total RNAs were contented with requirement of microarray; the quality control of the microarray assays was well performed; all microarray data were comparable; miRNAs aberrantly expressed in HBV cell models were screened by comparing all these data from microarrays. Furthermore, the results of microarrays were validated by a self-established stem-loop qRT-PCR. These results showed that the miRNAs aberrantly expressed in HBV cell models could be applied for target prediction.2. Target prediction of miRNAs related to replication of HBVIn this study, we preformed target prediction of miRNAs related to replication of HBV in HBV genome or human genome, with the aim to understand the regulation of miRNAs in the progression of HBV-HCC.(1) Target prediction of miRNAs related to replication of HBV in HBV genomeFor a better understanding of the function of miRNAs in regulating the expression of HBV genes, we screened miRNAs which were inversely correlated to replication of HBV, and then predicted the targets in HBV genome by a self-established bioinformatics method, namely array-miRTar. The results showed that 29 miRNAs were inversely correlated to replication of HBV. Among them, four miRNAs could target HBV genes. For example, miR-15b was a miRNA which was inversely correlated to replication level of HBV, and had a highly credible target located in the overlap region of S gene and P gene. These results suggested that miR-15b might downregulate the expression of S gene or P gene by directly targeting their coding region﹙CDS﹚.(2) Target prediction of miRNAs related to replication of HBV in human genomeFor a better understanding of the function of miRNA in regulating the expression of tumor-associated genes, we screened miRNAs whose expression level were directly correlated to replication of HBV, and then predicted the targets in human genome. To analyze expression level of target mRNAs, we profiled the aberrant expression of mRNAs in HBV cell models by cDNA microarrays. We found that miR-192 was a miRNA which was directly correlated to replication of HBV, and RB1 which expressed inversely correlated to replication of HBV was one of the most highly credible targets of miR-192. Taken together, these results suggested that HBV might inhibit expression of RB1, by promoting expression of miR-192.3. Functional study of miRNAs related to replication of HBV﹙miR-192﹚To investigate the molecular mechanism by which HBV induces expression of miR-192, we tried to identify which protein encoded by HBV could enhance the activity of potential pri-miR-192 promoter. The results showed that over-expression of HBx, but not other proteins, could strongly enhance the activity of pri-miR-192 promoter. Our results also showed that HBx could upregulate the level of miR-192 mature. Our study identified a conserved region which contains a hepatocyte nuclear factor-1α﹙HNF-1α﹚ binding site and demonstrated that the conserved region is the critical sequence of pri-miR-192 promoter. The further study showed that HBx enhance the activity of pri-miR-192 promoter activity by the HNF-1αbinding site. Taken together, these results suggested that HBx can promote the expression of miR-192 at transcriptional level by enhancing the activity of miR-192 prompter through HNF-1αbinding site.To investigate the molecular mechanism by which miR-192 inhibits expression of RB1, eukaryotic expression vector of miRNA and 3’UTR reporter vector system were constructed, and subjected them to analyze the post-transcription gene silencing﹙PTGS﹚of RB1 by miR-192. Results demonstrated that miR-192 decreased the relative luciferase activities with full-length 3’UTR of RB1 in dose-dependent manner, indicating that miR-192 can downregulate RB1 expression by targeting its 3’UTR.4. ConclusionOur research showed that the regulation of RB1 by miR-192 may closely relate to the progression of HBV-HCC through analyzing miRNAs’aberrant expression in HBV cell models. Furthermore, our research found that HBx can enhance the activity of miR-192 prompter through HNF-1αbinding site while miR-192 can induce the PTGS of RB1 by targeting its 3’UTR.