| Hepatitis B virus (HBV) is one of the most serious and prevalent health problems, affecting more than 2 billion people worldwide. Although highly effective vaccines against hepatitis B virus have been available since 1982, there are still more than 400 million chronic carriers,75% of whom reside in Asia Pacific region including China. Hepatitis cirrhosis and hepatocellular carcinoma (HCC) and other end-stage liver diseases which were caused by chronic HBV infection, have become a serious harm to our people’s lives and health. In recent years, new anti-HBV drugs are continuously available, but the effect is still not satisfactory, it still need to explore new anti-HBV drugs and methods. Then, it is significant to research silencing of USP18 in the antiviral activity of interferon against hepatitis B virus infection (CHB).Background:Interferon-a is approved as one of the main therapeutic treatments for chronic hepatitis B virus infection, but only a small number of patients achieve sustained virological response. The molecular mechanisms underlying IFN-a resistance in those patients who do not respond remain elusive. Previous work in our laboratory identified the pre-activation of IFN signaling leading to increased expression of a subset of interferon stimulated genes in the pretreatment liver tissues of chronic HBV infected patients correlated with treatment non-response.Aims:To construct three short hairpin RNA (shRNA) interference recombinant lentivirus-based vectors of human USP18 by RNA interference, and detect the strongest RNAi effect of USP18 shRNA sequence. After HepG2.2.15 cells were transfected recombinant lentiviral, HBVDNA were detected by diagnostic kit for quantification of hepatitis B virus DNA and the expression of ISG15 was detected by western blot.Methods:In this study chemically synthesized small interfering RNA (siRNA) sequences (siRNAl,siRNA2,siRNA3) targeting USP18 gene in vitro was transfected in HepG2.2.15 cells by lentivirus-based vectors (LV). The expression levels of USP 18 mRNA were detected by real time PCR (RT-PCR), and the inhibition of USP 18 which was the most significant was selected. While HepG2.2.15 cells were transfected by USP 18 RNAi recombinant lentiviral, these were divided into five groups such as A group, B group, C group, D group, E group (A group: PEG-IFNa+USP18-RNA-LV 1#, B group:USP18-RNA-LV 1#, C group:PEG-IFNa+NC-GFP-LV, D group:NC-GFP-LV, E group:blank group). Eight hours, three days, seven days after transfected, we collected cell supernatant and detected HBVDNA。Three days after transfected, the expression of ISG15 was detected by western blot.Results:We successfully constructed USP18 RNAi recombinant lentiviral(USP18-RNA-LV1#, USP18-RNA-LV2#,USP18-RNA-LV3#). Five days after HepG2.2.15 cells were transfected by USP18 RNAi recombinant lentiviral, RT-PCR results showed that the expression of USP18 mRNA of HepG2.2.15 cells were effectively more decreased by USP18-RNA-LV 1#, USP18-RNA-LV 2#, USP18-RNA-LV 3# than the control group (Negative group:NC-GFP-LV and blank group), and we successfully selected the effectively decreasest shRNAl sequences targeting USP18 gene (USP18-RNA-LV 1#)(P<0.05). Eight hours, three days, seven days after transfected, with the passage of time, the inhibitory effect was efficient, especially A group. And western blot results further confirmed the above results, a further verification of silencing USP18 has a certain significance at the protein level.Conclusion:The results indicate that the USP18 RNAi recombinant lentivirus vectors were successfully constructed, and silencing of USP18 potentiates the antiviral activity of interferon against hepatitis B virus infection, which will bring new hope for CHB. In the future, I believe that with the deepening of research in this field, the application of RNAi technology against HBV experiment will also toward a clinical application from an experimental study. |
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