DDX41蛋白通过活化IRF3抑制乙型肝炎病毒的复制

DDX41 reduces hepatitis B virus replication by activating IRF3 pathway

目的探讨含DEAD框解旋酶41(DDX41)基因对乙型肝炎病毒(HBV)复制能力的影响及其作用机制。方法通过慢病毒包装将稳定过表达DDX41或特异敲低DDX41的短发卡RNA(shRNA)重组质粒转染至HepG2.2.15细胞系,筛选和建立稳定过表达或敲低DDX41的细胞株;通过CRISPR-Cas9技术构建敲除DDX41的HepG2.2.15细胞株;蛋白免疫印迹法检测DDX41蛋白表达水平以及通路验证;酶联免疫吸附法(ELISA)检测乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)表达水平;实时荧光定量PCR(RT-PCR)检测HBV复制相关DNA、RNA以及干扰素mRNA表达水平;Northern印迹法检测HBV总RNA。结果过表达DDX41可显著抑制HepG2.2.15细胞系中HBV的复制;而敲低或敲除DDX41可显著促进HepG2.2.15细胞系中HBV的复制。过表达DDX41可显著促进干扰素调节因子3(IRF3)的磷酸化和干扰素β(IFN-β)的表达;而敲低或敲除DDX41可显著抑制IRF3的磷酸化和IFN-β的表达。结论在肝癌细胞系HepG2.2.15中,DDX41蛋白通过促进IRF3的磷酸化并诱导干扰素β的表达,进而发挥抑制HBV复制的功能。

Objective To investigate the effects of DEAD-box helicase 41(DDX41)on the replication of hepatitis B virus(HBV) in hepatoma cell line HepG2.2.15 and the underlying molecular mechanism. Methods Recombinant plasmid vectors for overexpression of DDX41 or containing short hairpin RNAs(shRNAs)targeting DDX41 were transfected into HepG2.2.15 cells by lentivirus packaging. CRISPR-Cas9 assays were performed to knock out DDX41 in HepG2.2.15.Western blotting assays were performed to detect the protein levels of DDX41 and verify the relevant pathways,enzymelinked immunosorbent assays(ELISA)were performed to detect the expression levels of HBsAg and HBeAg,real-time fluorescence quantitative PCR(RT-PCR)assays were used to detect the expression levels of HBV replication-related mRNA,and DNA and interferon mRNA. Northern blotting assays were performed to detect HBV total RNAs. Results Overexpression of DDX41 significantly inhibited HBV replication in HepG2.2.15 cells,whereas knockdown or knockout of DDX41 significantly promoted HBV replication. Mechanistically,overexpression of DDX41 significantly promoted phosphorylation of IRF3 and increased the expression levels of IFN-β without affecting the NF-κB pathway. Knockdown or knockout of DDX41 significantly reduced phosphorylation of IRF3 and downregulated the expression levels of IFN-β.Conclusion The DDX41 protein in the hepatocellular carcinoma cell line HepG2.2.15 may promote the phosphorylation of IRF3,which can lead to an increase in IFNβ and inhibit HBV replication.