Screening And Functional Study Of HBV Infection Related MicroRNAs

HBV infection is one of the most infectious diseases in China. There are about120million HBV carriers in our country, and among them20million people arechronic hepatitis patients. Hepatitis B virusinfection and correlated diseases have aserious effect on patient's health, and burden their family and society in economy.Now the mechanism of HBV pathogenesis is still not very clear, and there is still nospecific drug for therapy of HBV infection. Interferon and nucleic acid analogues arewidely used as anti-HBV drug clinically, but interferon has disadvantages ofgreat sideeffects, while nucleic acid analogues have disadvantages of relapse. Consequently,new ways are needed in new and efficient medicine development for HBV infection.microRNAs (miRNAs) are small noncoding RNAs which are widely encoded byplant, animal and viral gemomes. miRNAs play significant roles in a variety ofphysiological processes including cell growth, differentiation, apoptosis,tumorigenesis and virus pathogenesis. Recent researches suggest that miRNAs mayplay important role in the process of viral infection. Our research is to screen andvalidate HBV infection related miRNAs, and to study the functional and molecularmechanisms of them. We hope our research could provide theory basis for HBVinfection mechanism from the point of miRNA, and offer potential diagnosticbiomarker and drug target for diagnose and therapy of HBV correlated disease.This research screened an established hsa-miRNA expression library in the celltransient transfection model to identify miRNAs with the potential of interfering withHBV replication, and chose the representative miRNAs for molecular mechanismsresearch of anti-HBV. Our research contents include two aspects as follows:1)screened a hsa-miRNA expression library by HepG2transfection to identify miRNAswith the potential of interfering with HBV replication.2) molecular mechanismresearch of HBV replication inhibition by miR-141. The conclusions are as follow:1. The hsa-miRNA expression library was screened in the model of transient transfecting miRNA expression vector and HBV expression vector into HepG2cell,and identified six miRNAs related to HBV replication. HBsAg level detection of thecell culture after transfection indicated that miR-98and miR-137have somepromotive effects on HBsAg expression, whereas miR-101,miR-125a,miR-125b,miR-141have some inhibitive effects on HBsAg expression.2. The host target gene of the6miRNAs mentioned above were predicted inbioinformatics way, and the results contain several genes participated intranscriptional regulation or important signal pathway, including FOXA1, PPARA,RXRA as the essential cell regulatory factor in HBV replication. The interactionsbetween these miRNAs and their target genes were verified using luciferase reporter.The result indicated that miR-101has interaction with HNF1B3'-UTR, miR-137hasinteractions with3'-UTR of NF1and SP1, miR-141has interaction with PPARA3'-UTR. These three miRNAs could repress luciferase activities in the experiment.3. The mechanism research of HBV replication inhibition by miR-141. Wechose miR-141as the research object for HBV replication inhibition based on theresults above. In cell transient transfection experiment, synthesised miR-141(miR-141mimics) repressed HBs/HBe expression and HBV DNA replication, andmiR-141inhibitor promoted HBs/HBe expression. The target sites of miR-141inPPARA were predicted using the bioinformatics software (TargetScan). The resultindicated that there are four possible target sites of miR-141in PPARA3'-UTR.These sites were checked using luciferase assay, and it showed thatmiR-141couldinteract with three target sites in PPARA3'-UTR. The inhibition of HBs/HBeexpression and HBV DNA replication after PPARA repression were verified usingPPARA specific siRNAs, and this confirmed the possibility of HBV replicationinhibition by miR-141. The result of promoter functional assay using luciferasereporter indicated that inhibition of PPARA by miR-141could repress the activities ofHBV promoters, and this explained the molecular mechanism of miR-141inhibitiveeffect on HBV replication.In conclusion, our research screened a hsa-miRNA expression library in celltransient transfection model and miR-141was identified and checked to haveinhibitive effect on HBV replication, and PPARA was proved to be the target gene ofmiR-141in this process.