The HBV Infection Related ADAR1 Gene Function Inquiry

BackgroundsHepatitis B is a worldwide blood-borne infectious disease of the liver, which may develop into chronic liver disease and chronic infection.The patients have a high risk of dying of cirrhosis and liver cancer. Interferon a (IFNα) is one of the most commonly used clinical drugs against chronic hepatitis B (CHB), which can significantly inhibit the replication of HBV. Studies have confirmed that the antiviral activity is mediated by IFN a binding to cell surface receptors to activate the downstream antiviral proteins, such as adenosine deaminases acting on RNA (ADAR1) and myxovirus-resistant GTPase (MxA), through a series of signal transduction pathways. Association studies provide evidence that single nucleotide polymorphism (SNP) in the ADAR1 gene correlate with the response to IFN in the treatment of multiple sclerosis and chronic hepatitis C. In addition, our previous studies suggested that rs4845384 in ADAR1 to be associated with no response to IFN treatment in chronic hepatitis B patients. We have found that rs4845384 was correlated with HBeAg seroconversion and HBsAg seroclearance both spontaneously and interferon induced.ADAR1 is an important interferon-sitmulating gene in alpha interferon (IFNa) signaling pathway, which catalyzes adenosine to inosine of dsRNA substrates. At the same time, ADAR1 also functions as a RNA-binding protein independently from its editing activity. Studies showed that ADAR1 played an important role in the tumorigenesis and development. The expression of ADARl was regulated by endogenous regulators of miRNAs, resulting in the malignancy of tumor. In addition, it is revealed that ADAR1 exhibited proviral or antiviral effects according to different types of viral infections. Recently, several studies pointed out that ADAR1 can inhibit Hepatitis C virus replication and affect its activity, with a potential antiviral mechanism. Till date, its role and mechanism are controversial HBV infections.To preliminarily interprate the exact role of ADAR1 gene and its possible mechanism in HBV infection, dual luciferase reporter gene assay (DLR), real-time quantitative PCR assay, enzyme linked immunosorbent assay (ELISA) and other experimental methods were applied. In a word, this study is aimed to provide new targets and strategies based on ADAR1 for the research focus on the pathogenic mechanism of hepatitis B virus infection and the treatment of hepatitis B.MethodsDual luciferase reporter assay (in 7 cell lines:HeLa, SK-HEP-1, QGY-7703, HepG2, HepG2.2.15, Hep3B and SMMC-7721) was used to reveal the effect of rs4845384 on reporter gene activities; ADAR1 expression plasmids and hairpin plasimids were constructed and transiently transfected into HBV positive cell line HepG2.2.15 to detect supernatant HBV infection markers by ELISA and real-time quantitative PCR method, thus proving that ADAR1 played a role in HBV infection; The screening of miRNA interacted with ADAR1 gene 3’-UTR was conducted by the construction of ADAR1 gene 3’-UTR luciferase reporter plasmid, prediction of related miRNAs via websites (microRNA.org-Targets and Expression, TargetScan, miRTarBase), synthesis of miRNA expression plasmids and verification through DLR assays.Results1. The reporter gene activity of ADAR1 rs4845384We cloned genomic regions from nucleotide -318 to +95 bp around rs4845384, which were made into two reporters:pGL3-rs4845384A and pGL3-rs4845384G. In the vast majority of cells, pGL3-rs4845384G constructs exhibited higher luciferase expression compared to pGL3-rs4845384A constructs (P<0.05), especially in HBV positive cell line HepG2.2.15. Moreover, the luciferase expression was visibly elevated in most cases with the addtion of IFNa.2. In vitro functional study of ADAR1 associated with HBV infection2.1 Plasmid preparationOverexpressive plasmids of ADAR1 two configurations (p110, p150) were successfully constructed and the siRNAs specifically binding ADAR1 and interfering with its expression were screened and made into hairpin plasmids. After plasmids were transiently transfected into eukaryotic cell lines, such as HepG2.2.15, the overexpression and interference effect is proved good.2.2 The detection of supernatant HBV infection markers of HepG2.2.15Seventy two hours after transient transfection of related plasmid, the ELISA experiments found that overexpression of ADAR1 (p110/p150) supernatant HBsAg and HBeAg were significantly increased. (P<0.05) However, in the case of sh-ADAR1, supernatant HBsAg and HBeAg were significantly decreased (P<0.05); Extracting supernatant HBV DNA and real-time quantitative PCR showed that when ADAR1 is overexpressed, the secretion of HBV DNA significantly increased. (P<0.05) In contrast, if ADAR1 was down-regulated, the supernatant HBV DNA copis significantly declined. (P<0.05)3. Preliminary screening and verification of miRNA interacted with ADAR1 3’-UTRSeven miRNAs were proved to be interacted with ADAR1:hsa-miR-93, hsa-miR-143, hsa-miR-20b, hsa-miR-20a, hsa-miR-613, hsa-miR-106a and hsa-miR-206.Conclusion1. The luciferase studies suggest that rs4845384 may play a role in the regulation of ADAR1 gene expression.2. After overexpression or knockdown of ADAR1 in HepG2.2.15, HBV infection markers in the supernatant were significantly increased or decreased, so as to make a conclusion that ADAR1 gene may promote HBV virus replication and secretion.3. By preliminary screening of miRNA, it was found that hsa-miR-93, hsa-miR-143, hsa-miR-20b, hsa-miR-20a, hsa-miR-613, hsa-miR-106a and hsa-miR-206 could interact with ADAR1 3’-UTR.