Modulation Of Hepatitis B Virus Replication And Cellular Gene Expression By MicroRNA-1

Objective1.To explore the effects of miR-1 on HBV replication and gene expression in different cellular models in vitro.2.To define the possible mechanisms of how does miR-1 regulate HBV replication directly or indirectly,to screen and verify the potential target genes of miR-1,to examine the influence of miR-1 on hepatoma cell proliferation and differentiation.3.To study the potential role of the histone deacetylase(HDAC) gene family in the regulation of HBV replication by miRNA.Methods1.Chemical synthesized miR-1 mimic was transfected into HBV stable transfection cell lines HepG2.215 and HepG2.117,or co-transfected to Huh7 and HepG2 cells with the HBV infectious clone pSM or pHY106 which have replication capacity.Intracellular HBV replication intermediates were detected by southern blot.Intracellular HBV transcripts and HBV virions in the supernatant were detected by realtime PCR.The expression of HBsAg and HBeAg antigens were detected by CMIA test.The HBcAg antigen expression was detected by western blot.2.siDicer,mut-miR-1 and miR-206 were transfected into HBV stable transfection cell line HepG2.215.Intracellular HBV replication intermediates were detected by southern blot. The expression of HBsAg and HBeAg antigens were detected by CMIA.The miR-1 specific inhibitor(2'OMe-miR1),a set of siRNAs targeting silencing complex (siRCK/p54,siGW182,siAgo-2,siFxR1) were co-transfected with miR-1 into HepG2.215.Intracellular HBV replication intermediates were detected by southern blot. The expression of HBsAg and HBeAg antigens were detected by CMIA test.3.Bioinformatics software and luciferase reporter gene system were used to find the potential miR-1 binding site.Reporter plasmids containing S,C,X promoter sequences of HBV genome were cloned into pGL3-basic plasmid;containing fragments of HBV genome sequence and E2F5 gene 3 'non-coding region(3'UTR) were cloned into pmiR-report plasmid.These reporters were co-transfected with miR-1 or control miRNA into HepG2 and Huh7 cells,luciferase reporter gene expression were detected. Intracellular E2F5 protein expression was detected by western blot.4.The effects of miR-1 on HepG2.215 cell proliferation and cell cycle were detected by WST-1 cell proliferation assay,H3-thymidine incorporation assay and flow cytometry(PI staining).Cell differentiation-related genes Albumin and PEPCK expression were detected by realtime RT-PCR.5.The siRNA targeting the HDAC gene family(siHDAC1,siHDAC2,siHDAC3,siHDAC4) were transfected to HepG2.215 cells.Intracellular HBV replication intermediates were detected by southern blot.The expression of HBsAg and HBeAg antigens were detected by CMIA test.HDAC4 protein expression after miR-1 transfectionn was detected by western blot.Results1.Blockage of the cellular miRNA synthesis by siDicer can reduce the HBV replication. Ectopic miR-1 expression in HepG2.215 cells greatly enhanced HBV replication in time-dependent and dose-dependent manner.HBV transcripts,the expression of viral proteins(HBsAg,HBeAg,HBcAg) and the secreted virions in the supernatant were also increased significantly when compared with the control.Similar effects can be observed in other cell models,such as HepG2.117 cells and HBV replicon transiently transfected HepG2 and Huh7 cells.2.Mutanted miR-1(m-miR-1) with mutated seed sequence and miR-206,which share same seed sequence as miR-1 had no effect on HBV replication in HepG2.215 cells,miR-1 inhibitor,2-OMe-anti-miR1 can completely block the upregulation of HBV-replication by miR-1,siRNA targeting the silencing complex can reduce the HBV replication,and partially block the upregulation of HBV-replication by miR-1.3.miR-1 can enhance the transcription activities of HBV promoters,especially the S gene promoter,miR-1 can also upregulate the expression of pmiR-report plasmid containing full-length HBV genome sequence,as well as partial HBV genome sequence(1830-2849). However,no possible binding site was identified in the genome sequence of HBV by using miRNA target gene prediction software.4.Cell proliferation and DNA replication experiment suggested miR-1 transfection can inhibit the cell growth rate and slow down the cell division.Cell cycle analysis showed that miR-1 transfection can increase the number of G1 phase cells,and can lead to significant G1/S cycle block.Western blot showed the expression of HDAC4 and E2F5 protein were decreased by miR-1.5.siHDAC3 and siHDAC4 transfection can increase the HBV replication and gene expression,while siHDAC1 and siHDAC2 transfection have no obvious effects.HDAC chemical inhibitor TSA can increase the HBV replication and decrease the expression of HDAC4 protein.Realtime RT-PCR found that the expression of the cell differentiation-related gene Albumin and PEPCK were increased.Conclusions1.miR-1 can significantly promote the replication and gene expression,and increase the secretion of HBV through the transcription and post-transcriptional regulatory role. 2.miR-1 increases the replication of HBV in sequence-specific way with silencing complex involve in the process.3.miR-1 may block the cell cycle through its target gene E2F5 and HDAC4,inhibit cell proliferation and division,promot the differentiation of hepatoma carcinoma cells, thereby change the malignant phenotype of tumor cells.4.miR-1 is likely to increase the replication of HBV with a synergistic effect by regulating many types of viral replication-related gene expression.Significances of the study1.Our works provided the first evidence of the existence of cellular miRNA which can affect HBV replication,miR-1 regulate the expression of specific cellular genes expression in hepatocytes at sequence-dependent manner,and then regulate the HBV replication and gene expression.Our works lay a foundation for further study of the biological mechanism of HBV replication in liver cells,and provide a reference for miRNA biology functional study.2.Our works preliminarily described the possible molecular mechanisms of regulation of HBV replication by miR-1 by using sequence analysis and reporter gene technology, provide new ideas for further clarify the molecular mechanisms of HBV infection and discovery of new anti-HBV targeting molecules.3.Our study found that miR-1 target genes are important genes which mostly involve in the cell growth and apoptosis,meanwhile,we observed miRNA molecule could inhibit liver cancer cell proliferation,promote cell differentiation,suggesting that miRNA plays an important role in tumor suppression,can be applied for the gene therapy of liver cancer.