The Study On The Function And Molecular Mechanism Of MicroRNA-15b In HBV Replication

Hepatitis B virus(HBV) infection is a public health problem worldwide. HBV infection can cause either acute or chronic hepatitis B in infected individuals, even leading to hepatitis and hepatocellular carcinoma(HCC). After HBV infecting host, HBV covalently closed circular DNA(HBV ccc DNA) can incorporate into the host chromatin and replicate in host, inducing the maintenance of chronic HBV infection. For the treatment of HBV infection in clinical, α-interferon and nucleos(t)ide analogs(NA) could inhibit HBV replication to some extent, but because of the existence of drug-resistant HBV mutants and HBV ccc DNA, clinical drugs cannot induce persistent antivirus immune response and clear HBV in infected cells. In order to inhibit HBV replication and reduce pathogenicity in vivo, we need to study the interaction of HBV-host and the molecular mechanisms of HBV replication. Recent studies showed that micro RNA(mi RNA) is a new regulator of virus-host interaction and play a pivotal role in the replication and pathogenesis of virus. To further study the role of mi RNA in HBV replication and pathogenesis would help us to further elucidate the mechanisms of virus-host interaction and to provide new targets and strategies for the development of new anti-HBV drugs.To explore HBV replication-related mi RNAs, we first compared the mi RNA profile using different HBV replication level cell model by mi RNA microarray and found out HBV replication-related mi RNA. Then we study the interaction and molecular mechanisms between mi RNA and HBV replication and elucidate the role of mi RNA in the interaction of HBV-host. 1. Study on the effect of mi R-15 b on HBV replication and expression(1) Screening and identifying of HBV replication-related mi RNAWe used Hep G2, Hep G2.2.15 and Hep Ad38 as none HBV replication, low HBV replication and higher HBV replication cell model to compare the mi RNA profile by mi RNA microarray and select six HBV replication-related mi RNAs as candidates which decreased as HBV level increased. We then transfected the six mi RNA mimics into HBV transiently expressing cells to select HBV replication-related mi RNA by detecting HBe Ag expression. The results showed that mi R-15 b, but not other mi RNAs, could promote HBe Ag expression. So we select mi R-15 b as a candidate to study the role and the mechanisms of mi R-15 b in HBV replication.(2) The effect of mi R-15 b on HBV replication and expressionWe analyzed the effect of mi R-15 b on HBV replication and expression by detecting the extracellular HBe Ag, HBs Ag, HBV DNA copy number and the intracellular HBV RNA, HBV DNA replicative intermediates. The results showed that overexpressed mi R-15 b level promoted HBV replication and expression, while decreased mi R-15 b level inhibited HBV replication and expression in a persistent HBV expression cell line Hep G2.2.15 and in a transiently HBV expression cell line Huh7. In order to study the effect of mi R-15 b on HBV expression and replication in vivo, we detected the HBV DNA copy number in mouse model of chronic HBV infection mediated by recombinant adeno-associated virus 8 through hydrodynamic injection of mi R-15 b inhibitor and the results showed that mi R-15 b downregulation inhibited HBV DNA copy number in mice serum. All the results demonstrated that mi R-15 b could promote HBV replication and expression. 2. The study on the molecular mechanism of mi R-15 b in the regulation of HBV replication and expression(1) To study on whether mi R-15 b directly target HBV transcripts to regulate HBV expression and replicationCellular mi RNAs are able to regulate HBV either by a directly binding to HBV transcripts, or by targeting to cellular transcriptions factors required for HBV transcription and replication. We first analyzed whether mi R-15 b could directly target HBV transcripts by bioinformatics and the results showed that there may be a putative mi R-15 b binding sites in HBV genome(nt 417-423). We cloned the potential target sits into mi RNA target gene validating vector and transfected with mi R-15 b mimics. The dual-luciferase reporter assay results showed that mi R-15 b did not influence the activity of luciferase reporter which means that mi R-15 b could not directly target HBV transcripts to regulate HBV replication.(2) To study on whether mi R-15 b target HBV replication-related factors to regulate HBV expression and replicationAfter excluding the possibility of mi R-15 b directly targeting HBV transcripts, we assume that mi R-15 b may target to HBV replication-related factors to regulate HBV expression and replication. Formation and transcription of HBV ccc DNA is an important step during HBV replication. HBV promoters, enhancers and transcription factors play a pivotal role during HBV transcription. After excluding the possibility of mi R-15 b directly targeting HBV transcripts, we supposed that mi R-15 b may regulate HBV replication and expression by influencing HBV promoters or enhancers through targeting HBV transcription-related factors. We study the effect of mi R-15 b on HBV promoters or enhancers. We cloned all HBV promoters and enhancers into luciferase reporter p GL3-Basic and transfected them with mi R-15 b mimics into cells,the dual-luciferase reporter assay results showed that mi R-15 b could only promote the activity of HBV EnhancerⅠ, but not other promoters or enhancers. We assume that mi R-15 b may target HBV EnhancerⅠtranscription-related factors to regulate EnhancerⅠ activity. We analyzed the mediators by informatics which should be the target of mi R-15 b and could bind to HBV EnhancerⅠ. Prediction showed that hepatocyte nuclear factor 1α(HNF1α) maybe a target of mi R-15 b and conld bind to HBV EnhancerⅠwhich means that HNF1α is the promising mediators of mi R-15 b promoting HBV EnhancerⅠactivity.(3) Validation of mi R-15 b targetTo test whether mi R-15 b interacts directly with HNF1α, we cloned the wild-type and mutant HNF1α 3’-UTR into the mi RNA target gene validating vector, and transfected them with mi R-15 b mimics into cells, the dual-luciferase reporter assay results showed that addition of mi R-15 b mimics resulted in a significant decrease in activity for the reporter carrying the wildtype HNF1α 3’-UTR, but did not affect the reporter activity with the mutant HNF1α 3’-UTR which means that mi R-15 b could directly target HNF1α 3’-UTR. To further study the effect of mi R-15 b on HNF1α m RNA and protein in cells, we found that mi R-15 b overexpression inhibit the expression of HNF1α m RNA and protein. Inversely, inhibition of endogenous mi R-15 b resulted in the elevated expression of HNF1α m RNA and protein. A similar phenomenon was also observed in vivo. These above results demonstrated that HNF1α is the target of mi R-15 b.(4) Study on the role of HNF1α in the regulation of HBV replication and expressionBioinformatics analysis predicted that there was a putative HNF1α binding sites in HBV EnhancerⅠ. In order to validate this, we cloned the wild-type and mutant HBV EnhancerⅠinto luciferase reporter p GL3-basic and transfected them with HNF1α expression vector into cells, the dual-luciferase reporter assay results showed that addition of HNF1α resulted in a significant decrease in activity for the reporter carrying the wildtype HBV EnhancerⅠ, but did not affect the reporter activity with the mutant HBV EnhancerⅠwhich means that HNF1α could directly target HBV Enhancer Ⅰ.To further study the effect of HNF1α on HBV replication and expression, we found that HBV replication and expression were decreased if HNF1α was increased, but increased if HNF1α was decreased. These above results demonstrated that HNF1α can directly target HBV EnhancerⅠand inhibit HBV replication and expression by decreasing HBV EnhancerⅠactivity.(5) HNF1α is a mediator of mi R-15 b promoting HBV replication and expressionThe above results showed that HNF1α is a target of mi R-15 b, meanwhile, HNF1α could inhibit HBV replication through decreasing HBV EnhancerⅠactivity. So we assumed that mi R-15 b could promote HBV replication and expression by targeting HNF1α, a negative regulator of HBV Enhancer I. To validate our assume, we tested the role of HNF1α in mi R-15 b promoting HBV EnhancerⅠactivity. The dual-luciferase reporter assay results showed that increasing mi R-15 b level in the system increased HBV Enhancer I activity, but further addition of HNF1α almost abolished the increase brought by mi R-15 b. Similarly, in p HBV1.2 transfected Huh7 cells, addition of mi R-15 b level increased both HBe Ag and HBs Ag expression, while overexpression of HNF1α abolished the promotion of HBV antigen brought by mi R-15 b. These above results demonstrated that HNF1α is the mediator of mi R-15 b promoting HBV Enhancer I activity and promoting HBV replication and expression. 3. Study on the effect of HBV infection on mi R-15 b expressionOur mi RNA microarray and previous experiments results showed that mi R-15 b decreased as HBV level increased. In order to study whether HBV can regulate mi R-15 b expression, we detect the effect of HBV replication and expression on mi R-15 b expression and found that HBV could inhibit mi R-15 b expression in vitro and in vivo. To further determine which protein encoded by four HBV genes correlates with mi R-15 b repression, we examined the expression of mi R-15 b in cells transiently expressing individual HBV genes, and found that mi R-15 b expression was significantly lower in cells transfected with HBx, but not with other HBV genes. We further study the kinetic expression of mi R-15 b in the livers of HBx transgenic mice and found that mi R-15 b decreased with time. These above results demonstrated that HBV x protein inhibit mi R-15 b expression in host cells.In this study, we demonstrated that mi R-15 b promotes HBV replication by targeting HNF1α, a negative regulator of HBV Enhancer I, and mi R-15 b expression in hepatocytes is down-regulated by HBV, and more specifically, by HBx. We believe that there was a negative feedback regulation between mi R-15 b and HBV. HBV can escape host immune surveillance through negative regulating HBV replication by inhibiting mi R-15 b, a promoter of HBV. The reciprocal regulation between mi R-15 b and HBV may help to control the level of HBV replication and play a role in the maintenance of chronic HBV infection.