Mechanism Study Of ADARs Downregulated Mavs By Editing Its 3'UTR To Influence Hepatitis B Virus Expression

ObjectiveAccording to statistics,the number of chronic hepatitis B virus carriers in the world is very large,more than 350 million people,and in the Chinese estimated about 130 million hepatitis B virus carriers.Chronic HBV infection has been a serious threat to the health of people.After infection with HBV,about 10%patients who develop chronic infection,virus persistent replication can cause liver cell damage,necrosis,degeneration,15%?40%will further develop into cirrhosis and liver cancer.Therefore,HBV infection caused by chronic hepatitis B further lead to cirrhosis and liver cancer is an important model for our nation of hepatocellular carcinoma.The study showed that viral factors,the interaction of genetic factors environmental factors and host,affect the outcome of HBV infection.Therefore,it is so importance to study the mechanism of HBV replication expression relative regulatory genes and therapeutic approaches.This study preliminarily explores the influence of the ADAR family on the replication of hepatitis B virus by RNA editing and altering the expression of the organism and viral genes.MethodsIn this study,using transcriptome sequencing,sanger sequencing and pyrosequencing analysis method to screen the non SNP,A-to-G editing sites in the 3'UTR of the mitochondrial antiviral protein gene by ADAR1 and ADAR2;To construct thedual luciferase plasmid of MAVS gene 3 'UTR,and to investigate the effect of the RNA editing function on the 3'UTR to the expression of gene;Further explore the relationship between ADAR family and MAVS from mRNA and protein level;Then establish CDS of the editing gene overexpression plasmid and sh-RNA knockdown plasmid,and transfected into HepG2.2.15 liver tumor cell lines,containing HBV whole genome can expression HBV antigen.ELISA,real-time fluorescent quantitative PCR and Western blotting method to detect HBV markers(HBsAg,HBeAg,HBV DNA,HBV cccDNA,3.5 kb HBV RNA,total HBV RNA and HB c)in the supernatant and cells,verify whether the role of this gene in HBV infection.Results1.The sequencing results showed that the chr20:3869744 and chr20:3870562 sites of MAVS might be the RNA editing sites of ADAR1 and ADAR2;2.The dual luciferase reporter gene assay showed that the expression of 3'UTR in the normal A allele of MAVS gene was significantly higher than that of G(P<0.05),especially in HBV positive cells HepG2.2.15;3.The protein level and mRNA level showed that the expression of ADAR1 and ADAR2 gene decreased the expression of MAVS(P<0.05);4.The effect of MAVS on the expression of HBV showed that the expression of HBV in the supernatant and intracellular was significantly decreased after the up regulation of MAVS expression(P<0.05),and the expression of HBV markers was increased significantly after knockdown the expression of MAVS(P<0.05);5.Co-transfection of ADAR1 and MAVS overexpression and knockdown plasmid were found that after co-overexpressed,the intracellular and supernatant of HBV markers were significantly reduced(P<0.05);common knockdown expression,the markers of HBV have no significant difference in the expression;6.The protein levels results showed that the addition of interferon alpha in HBV positive HepG2.2.15 cells,the expression of MAVS decreased significantly(P<0.05);and applying and also increased the expression of MAVS,the HBV marker expression in the supernatant and intracellular decreased significantly then only IFN alpha(P<0.05).Conclusion1.transcriptome sequencing and pyrosequencing verification indicated that MAVS gene may be regulated by RNA editing of ADARl and ADAR2;2.The dual luciferase reporter assay suggest that the chr20:3869744 and chr20:3870562 sites of MAVS were edited to decrease the gene expression activity;3.The expression of HBV markers showed that the expression of MAVS may inhibit the replication and expression of HBV;4.The results of protein and mRNA suggest that IFN alpha and ADAR1 may inhibit the expression of mitochondrial antiviral protein MAVS,and thus affect the replication of HBV virus;5.The application of IFN a and enhance the expression of MAVS,can get better antiviral effect,which provides a potential new treatment strategy for MAVS based on IFN alpha,which has a certain clinical application value.6.In summary,the ADAR family,by editing the body's MAVS RNA,altered the expression of the MAVS gene,thereby inhibiting the replication of the hepatitis B virus.