The Roles And Regulations Of USP18 In The Anti-HBV Mechanisms Of Interferon Alpha

Chronic hepatitis B(CHB)is a prevalent health problem worldwide.Although highly effective vaccines against hepatitis B virus(HBV)are available,HBV infection remains to be one of the most serious health problems and there are still more than 400 million chronic carriers in the world.Chronic HBV infection can lead to cirrhosis or hepatocellular carcinoma and other serious end-stage liver diseases.Although various anti-HBV drugs emerging nowadays,interferon alpha(IFN-?)is still approved as one of the main choices in the treatment for HBV.IFN-? has such advantages as fixed course of treatment,low relapse rate after stoppong drug,and even the seroconversion of HBs Ag in some patients.The long acting PEG-IFN-? also has such advantages as long time interval and relatively few side effects.However,the sustained virological response in CHB patients is still unsatisfactory.The specific molecular mechanism of IFN-? resistance remains elusive.In our previous work,we identified that pre-activation of IFN-? signaling led to diffential expression of a subset of interferon-stimulated genes(ISGs)and immune-related genes in the pre-treatment liver tissues of treatmen responders and non-responders using Aglient Whole Human Genome Oligo Microarray.Among the differentially expressed genes,USP18 and isg15 are located in the same signal transduction pathway.USP18 was increased in tissue of non-responders,indicating that it may be related with the anti-HBV activity of IFN-?.Objective: To study the expression profile of USP18 in different hepatocellular carcinoma cell lines.To explore the effect of HBV infection of the expression of USP18 in hepatocellular carcinoma cells.To consetrcut the lentiviral vector sh RNA-USP18 and study its effect on the HBV replication in Hepg2.2.15 cells.Treat the Hepg2.2.15 cells with different dose of IFN-? and then to explore the effect of lentiviral vector sh RNA-USP18 on HBV replication.To explore the relationship between JAK/STAT signaling pathway and the sh RNA-USP18.To study the differentially expressed genes and the related biological process and pathways in Hepg2.2.15-sh RNA-USP18 using bioinformatics analysis.Methods: The m RNA level of USP18 in different hepatocellular carcinoma cell lines was measured using q RT-PCR.After transfection of HBV expression plasmid,the effect of HBV on the expression of USP18 m RNA and protein were measured using q RT-PCR and westen blot respectively,and the activity of USP18 promoter was assessed using dual luciferase reporter gene experiment.After construction of lentiviral vectors,the most effective one for USP18 interference was selected using q RT-PCR.The stable cell line was selected using puromycin after transduction of sh RNA-USP18.And then the efficiency of sh RNA-USP18 was measured using q RT-PCR and westen blot.After transduction of sh RNA-USP18,the expression of intracellular HBV DNA,pg RNA and total RNA was assessed using q RT-PCR.The secretion of HBs Ag and HBe Ag in the superenant was assessed using ELISA.After transfection of pc DNA3.1-USP18,the expression of intracellular HBV DNA,pg RNA and total RNA was assessed using q RT-PCR.The secretion of HBs Ag and HBe Ag in the superenant was assessed using ELISA.After transduction of sh RNA-USP18 and treating the Hepg2.2.15 cells with different dose of IFN-?,the expression of intracellular HBV DNA,pg RNA and total RNA was assessed using q RT-PCR.The secretion of HBs Ag and HBe Ag in the superenant was assessed using ELISA.The expression of intracellular HBc Ag was assessed using westen blot.After transduction of sh RNA-USP18 and treating the Hepg2.2.15 cells with IFN-?,the expression of ISG15,Mx A and IFIT1 m RNA and protein were measured using q RT-PCR and westen blot respectively.The expression of p-STAT1 and STAT1 after different time interval and different method of administration were measured using westen blot.After tranduction of sh RNA-USP18,the differentially expressed genes in Hepg2.2.15 cells were screened out using microarray.And the bioinformatics analysis for the differentially expressed genes was performed using public databases and softwares.Results: The expression level of USP18 in Hepg2.2.15 cells was higher than that of other hepatocellular carcinoma cell lines.Infection of HBV could promote the expression of USP18 m RNA and protein and increase the promoter activity.We have screened out the most effective sh RNA-USP18 lentiviral vector and have constructed the stable cell line Hepg2.2.15-sh RNA-USP18.The replication and secretion of HBV in Hepg2.2.15-sh RNA-USP18 cells were suppressed.Overexpression of USP18 could rescue this effect.After transduction of sh RNA-USP18,the anti-HBV activity of IFN-? was enchanced and the expression of ISGs was increased.The expression of p-STAT1 was increased and prolonged.The differentially expressed genes,the related biological process and signaing pathways in Hepg2.2.15-sh RNA-USP18 cells were screened out and analyzed preliminarily.Conclusion: The infection of HBV in Hep G2 cells could promote the expression of USP18 and enchance the activity of its promoter.We have constructed the most effective USP18 lentiviral vectors for interference and the replication of HBV was suppressed in Hepg2.2.15-sh RNA-USP18 cells.The anti-HBV activity of IFN-? was enchanced after transduction of sh RNA-USP18.USP18 can regulate the anti-HBV activity of IFN-? via regulating the JAK/STAT signaling pathway.