Mechanism Of MiR-146a Regulates Hepatitis B Virus Gene Expression And Replication

Objective: Hepatitis B virus(HBV)constantly generates infection and induces the abnormal expression of miR-146 a.However,currently more researches about miR-146 a targeting the key molecules of immune system including TRAF6,IRAK1 and other genes to control negatively the immune system.But,miR-146 a how to play its potential molecular mechanism in the process of HBV infection has not been fully revealed,especially in the study of type I interferon signaling pathway.This paper is trying to find a novel target gene of miR-146 a in the type I interferon signaling pathway to reveal the new mechanism of miR-146 a regulation the Hepatitis B virus gene expression and replication.Methods: Firstly,RT-qPCR was used to determine the expression of miR-146 a in p HBV1.3-transfected Huh7 and stable HBV-replicating hepatocytes cells.Then,we respectively transfect miR-146 a mimics and inhibitors to intervene endogenous miR-146 a expression.Whereafter,RT-q PCR and ELISA were used to detect HBV replication and expression in the above two cell models.Next,we used Vi Ta,miRandad bioinformatics software to predict the targeted relationship between miR-146 a and HBV transcript and related genes in the type I interferon signaling pathway.We then successfully inserted the candidate genes into the pmir GLO luciferase reporter vector for subsequent experiments.Finally,we successfully identified the SETD2 is one of miR-146 a target genes via luciferase assay.And SETD2 is deemed as the major object in this study.Furthermore,after interfering with the expression of endogenous miR-146 a,the expression of SETD2 gene and protein was detected respectively by RT-q PCR and Western Blot.Next,via overexpressing SETD2 and then treating with IFN-α for 6hours,the expression of HBV replication and expression level was detected respectively by RTq PCR and ELISA.Lastly,we used a rescue assay to investigate the positive effect of miR-146 a on HBV replication and transcription level if via negative regulation of the SETD2 gene.Results: We found that miR-146 a expression level was markedly up-regulated in p HBV1.3-transfected Huh7 and stable HBV-replicating hepatocytes cells.Meanwhile,we also found that overexpression of miR-146 a can promote HBV replication,when intervention endogenous expression of miR-146 a.Subsequently,the bioinformatics website predicted that there was no predictive correlation between miR-146 a and HBV transcript,we also successfully screened lnterferon-stimulated related gene such as RNase L,and SETD2.Next,we performed a dual luciferase reporter assay in HEK239 T cells.The results showed that miR-146 a could target directly SETD2 3’-UTR,whileRNASEL seem to no obvious targeting effect.We have also provided evidence that miR-146 a targeting SETD2-3’UTR in a specific site.Furthermore,we verified that overexpression of SETD2 and then IFN-α stimulation attenuates levels of HBV replication and expression in Hep G2.215 and p HBV1.3-transfected Huh7.Finally,we further proved that down-regulation of SETD2 by miR-146 a could attenuate HBV replication levels by rescue experiments.Conlusion: This study has concluded that miR-146 a expression can be up-regulated in HBV-infectd hepatocytes cells,and its up-regulation can promote the replication of HBV.It was confirmed that miR-146 a could directly target SETD2-3’ UTR region and negatively regulate the gene and protein expression of SETD2 by bioinformatics,Dual-luciferase assay,RT-q PCR and Western Blot.Secondly,we also confirmed that the overexpression of SETD2 can enhance the phosphorylation of STAT1 and the level of interferon-induced genes such as ISG15,RNASEL.Therefore,HBV replication can be inhibited to some extent.Finally,the potential mechanism that miR-146 a promotes the replication and expression of HBV through negative regulation of SETD2 gene is revealed by rescue experiments.This study thesis reveals the new mechanisms of miR-146 a in promoting HBV replication and expression,suggesting that miR-146 a may be a new breakthrough in the treatment of chronic HBV infection.