Regulation Of The Replication Of Hepatitis B Virus By MicroRNA

Objective:Chronic hepatitis B virus(HBV) infection is one of the leading cause of liver cirrhosis and hepatocellular carcinoma(HCC).Current strategies of HBV infection have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. MicroRNAs(miRNAs) are single-stranded noncoding RNAs of18to25nucleotides that play critical roles in a wide spectrum of biological processes. Accumulating evidence suggests that the miRNAs pathway also controls the replication of both RNA and DNA viruses. To address whether the miRNAs-silencing machinery influences HBV replication and antigen expression, miRNAs which can target crucial HBV genes including HBsAg or SP2genes or target cell genes including PRKCH and ATP5B genes, which play important roles in HBV infection, were studied.Methods:To address which miRNAs influences HBsAg expression, HBeAg expression or the proliferation of HBV producing HepG22.2.15cells, antisense oligonucleotide of special miRNAs was used to reduce the expression of endogenous miRNAs. On the basis of enzyme linked immunosorbent assay (ELISA) and CellTiter96(?) Aqueous Non-Radioactive Cell Proliferation Assay (MTS) performed on culture supernatant harvested from HepG22.2.15cells and HepG22.2.15cells72hours after transfection with miRNA ASOs, three miRNAs that inhibited HBsAg expression and one that enhanced HBsAg expression were selected.To understand the function of these miRNAs in HBV replication, culture supernatant harvested from HepG22.2.15cells transfected with miRNA ASOs and control oligo, respectively, HBV replication was tested by quantification of Hepatitis B virus DNA.To further analyze the mechanisms of miRNAs in controlling HBsAg expression and HBV replication, we used five well-established microRNA target prediction software-miRanda, TargetScan, PicTar, miRDB and ViTa were used to predict targets for the four miRNAs. Sequence analysis indicates that two of them do not directly target the HBV genome, which suggests that they might affect HBV replication by targeting cellular protein(s).Then, combined mRNA microarray with semi-quantitative RT-PCR was used to predict the target genes of these two miRNAs.These prediction results were conformed by using reporter vectors with the intact putative miRNAs recognition sequence from HBV or the3’-UTR of predicted gene (pcDNA3/EGFP-UTR) or randon mutations (pcDNA3/EGFP-UTRmut) cloned downstream of the EGFP gene, respectively. HEK293and HepG22.2.15were cotransfected with pcDNA3/EGFP-UTR with or without precursor miRNAs, with or without miRNA ASOs, respectively. So did the pcDNA3/EGFP-UTRmut. GFP and RFP activity were measured with a fluorescence spectrophotometer (HITACHI F4500)48hours after transfection. Results were represented as normalized ratio of EGFP to RFP.Results:First, those miRNAs that influenced HBsAg expression by ELISA and MTS were screened. There are three miRNAs, miR-199a-3p, miR-210and miR-185that inhibited the expression of HBsAg and miR-370enhanced HBsAg expression.Second, quantification of Hepatitis B virus DNA in culture supersnatant of HBV producing HepG22.2.15cells transfected with ASO indicates that miR-199a-3p, miR-210, miR-185reduce HBV production, compared with control ASO(Lacz) transfected cells.Third, microRNA target prediction software was used to screen for potential targets of miR-199a-3p, miR-210,miR-185and miR-370. Sequence analysis indicates that miR-199a-3p and miR-210might target HBsAg and SP2, respectively. Nevertheless, sequence analysis indicates that miR-185and miR-370do not directly target the viral genome, which suggests that they might affect HBV replication by targeting cellular protein(s). So, a7,267-element human cDNA microarray was used to screen up-regulated genes owning to miR-185or miR-370being blocked down. Combining target prediction and expression profiling, three candidate miR-185-targeted genes, PRKCH, ATP5B and NR1D1and two putative target genes including FUS and TPM4for miR-370were identified.Fourth, HBsAg, SP2and PRKCH were verified as targets for miR-199a-3p,miR-210and miR-185by EGFP reporter contructs, respectively.Conclusion:Up to date, this is the first report about human miRNAs that can inhibit HBV replication by targeting important HBV genes or cellular proteins. Evidence for an intricate physiological interplay between the cell’s miRNAs and HBV was provided. It offers an immense opportunity not only to understand the intricacies of host-pathogen interactions, and possible explanations to viral tropism, lantency and oncogenesis, but also develop novel biomarkers and therapeutics.