Study On The Mechanism Of Type ? Interferon And HBV In Regulating APOBEC3G Expression

Objective:Hepatitis B virus(HBV)belongs to the hepatotropic DNA virus family,HBV infection not only can cause acute hepatitis,but also can cause chronic hepatitis,cirrhosis,and even liver cancer.HBV may infect humans through many ways,lead to the spread of liver disease,which is seriously deleterious to human life and health.Interferon is one of the first-line drugs for the treatment of chronic hepatitis B.Interferon can induce hundreds of interferon stimulated genes(IFN Stimulated Genes,ISGs)expression including APOBEC3G by activating JAK-STAT signaling pathways.It was reported that type I interferon induced APOBEC3G expression did not depend on STAT1,however,the mechanism of type I interferon induced APOBEC3G expression remains unclear.APOBEC3G(Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G)is a kind of RNA/DNA editing enzyme,has cytidine deaminase activity,can make the cytosine(C)deaminate into uracil(U),which causes base mutations in virus genome,terminates the entire replication cycle.A research has shown that APOBEC3G can inhibit HBV replication in the hepatocyte,and the expression of APOBEC3G is restrained in the patients with HBV infection.However,the mechanism of regulating APOBEC3G by HBV has not been reported.Therefore,this topic will study the effect and mechanism of STAT1/3 in the process of type I interferon(IFN-?)induced APOBEC3G expression,and investigate the mechanism of regulating APOBEC3G by HBV.Methods:In order to verify APOBEC3G could be induced by type I interferon in the liver cell,HepG2 cells were stimulated by IFN?/?,Western Blot and q PCR were used to detect the expression of APOBEC3G in both the protein and mRNA level.Whereas type I interferon induced APOBEC3G expression is not dependent on STAT1,However,the mechanism of type I interferon induced APOBEC3G expression is still unclear,In order to further study the mechanism of type I interferon induced APOBEC3G gene expression,CRISPR/Cas9 technology was used to construct the cell line of STAT3 gene knockout in HepG2 cells,then STAT1,STAT3 knockout cells were stimulated by IFN?,Western Blot was used to detect the protein expression level of APOBEC3G;On the other hand,IFN? together with STAT3 inhibitor were added to stimulate HepG2 cells,Western Blot was used to detect the phosphorylation level of STAT3 and the APOBEC3G protein expression levels,which virified the effect of STAT3 in the process of type I interferon induced APOBEC3G gene expression.In this experiment,we verified the inhibitory effect of APOBEC3G on HBV,and constructed the eukaryotic expression vector of APOBEC3G by molecular cloning experiments,APOBEC3G expression vector and HBV expression plasmid were transfected to HepG2 cells by the liposome transfection,after transfection,ELISA was used to detect the levels of HBe Ag and HBs Ag,q PCR was used to detect the levels of HBV DNA and pg RNA.In the study of the regulation mechanism of HBV on the expression of APOBEC3G,first of all,blood samples were collected from 20 cases of patients with HBV infection and 20 normal people,serum and PBMC were isolated from whole blood,RNA was extracted from PBMC and reversed transcription to c DNA,q PCR was used to detect the mRNA level of APOBEC3G;Then,in order to further prove which virus factor in HBV can inhibit the expression of A3 G.Respectively the HepG2.2.15 cell culture supernatant and the serum of HBV infection patients was added to stimulate HepG2 cells together with IFN?,Western Blot and q PCR were used to detect the protein expression level and the mRNA level of APOBEC3G in the cell,Finally,HBs Ag recombinant protein was used to further verify our result,use HBs Ag recombinant protein stimulate HepG2 cells together with IFN?,then Western Blot was used to detect the phosphorylation level of STAT3 and APOBEC3G protein expression level,q PCR was used to detect the mRNA level of APOBEC3G.Results:Type I interferon can obviously increase APOBEC3G gene expression in HepG2 cells.After treatment with STAT3 inhibitor,the expression of APOBEC3G was obviously decreased.In STAT1 KO 293 T cells,the induction of APOBEC3G is not affected before 8 hours stimulation,but in STAT3 KO 293 T and HepG2 cells,the induction of APOBEC3G was obviously inhibited,And basal levels were also decreased.After the APOBEC3G and HBV expression plasmid were co-transfected into HepG2 cell,the levels of HBe Ag,HBs Ag,HBV DNA and pg RNA were obviously decreased.HBV can inhibit the expression of APOBEC3G gene,The expression of APOBEC3G gene in the patients with HBV infection is lower than uninfected normal person;After the stimulation by HepG2.2.15 cell culture supernatant and serum of HBV infection patients,the expression of APOBEC3G gene in cells was obviously decreased;After HBs Ag stimulation,the phosphorylation level of STAT3-Ser727?the protein expression level and mRNA level of APOBEC3G were significantly decreased,but the phosphorylation level of STAT3-Tyr705 was not affected.Conclusion:The induction of APOBEC3G by type I interferon was STAT3 dependent;HBs Ag inhibits the phosphorylation of STAT3-Ser727,which will further inhibit the process of type I interferon induced APOBEC3G gene expression and inhibit the antivirus effort of Type I IFN.