乙型肝炎病毒HBx蛋白诱导肝细胞泛素连接酶RNF5抑制STING/IFN-β信号通路的研究  

Hepatitis B virus X protein inhibits STING/IFN-βsignaling pathway through up-regulation of ubiquitin ligase RNF5 in hepatocytes

作　　者：杨凯 潘颖 宇芙蓉[1] 张发苏 陈谨[1] 李芳 YANG Kai;PAN Ying;YU Fu-rong;ZHANG Fa-Su;CHEN Jin;LI Fang(Department of Medical Technology,Anhui Medical College,Hefei 230601,China)

机构地区：[1]安徽医学高等专科学校医学技术学院,安徽合肥230601

出　　处：《中国病原生物学杂志》2022年第9期1005-1009,1014,共6页Journal of Pathogen Biology

基　　金：安徽省自然科学基金项目(No.2108085MH301);安徽省高校自然科学研究重大项目(No.KJ2020ZD68)。

摘　　要：目的研究乙型肝炎病毒编码X蛋白(HBx)对HepG2细胞STING/IFN-β信号通路的影响及其可能存在的机制。方法将含HBV全基因组1.5倍复制子质粒pHBV-48和缺失HBV X基因的HBV复制子质粒pHBV-48-X_(0)分别转染至HepG2细胞,采用细胞免疫荧光染色法观察细胞内HBx蛋白表达情况,同时采用实时荧光定量PCR和Western blot检测细胞内干扰素基因刺激因子(STING)基因表达水平及蛋白含量。分别转染空载体pEGFP-N1和HBV编码X基因表达质粒pEGFP-HBx至HepG2细胞,采用实时荧光定量PCR和Western blot检测细胞内STING及环指蛋白5(RNF5)基因和蛋白含量。采用基因干扰技术减少转染质粒的HepG2细胞RNF5表达,Western blot检测细胞STING、干扰素β(IFN-β)蛋白表达情况;以不同浓度蛋白酶抑制剂MG132处理转染pEGFP-HBx的HepG2细胞,Western blot检测细胞内STING、IFN-β蛋白表达情况。结果转染空载体、pHBV-48及pHBV-48-X_(0)的HepG2细胞STING基因相对含量差异无统计学意义(P>0.05);转染空载体的HepG2细胞STING蛋白(1.14±0.11)相对表达水平较转染pHBV-48的HepG2细胞(0.47±0.09)显著升高(P<0.01),但低于转染pHBV-48-X_(0)的HepG2细胞(2.15±0.18)(P<0.01)。转染空载体及pEGFP-HBx的HepG2细胞STING基因相对含量差异均无统计学意义(均P>0.05),但转染pEGFP-HBx的HepG2细胞STING蛋白(0.48±0.16)相对含量较转染空载体的细胞(0.87±0.22)显著降低(P<0.01);与转染空载体的HepG2细胞相比,转染pEGFP-HBx的HepG2细胞RNF5基因相对表达水平及蛋白相对含量均显著升高(均P<0.01)。减少HepG2细胞RNF5表达后,细胞内STING和IFN-β相对蛋白表达水平均显著升高(P<0.05或P<0.01)。随着蛋白酶抑制剂MG132浓度逐渐升高,表达HBx的HepG2细胞内STING和IFN-β蛋白相对含量逐渐上升。结论乙型肝炎病毒可能通过病毒编码蛋白HBx上调肝细胞泛素连接酶RNF5进而抑制STING/IFN-β信号通路。Objective To investigate the effect of hepatitis B virus-encoded X protein(HBx)on STING/IFN-βsignaling pathway in HepG2 cells and its possible mechanisms.Methods The HBV replicon plasmid pHBV-48 containing the 1.5-fold HBV genome and the HBV replicon plasmid pHBV-48-X0 lacking the HBV X gene were transfected into HepG2 cells,and the expression of Western blot.By transfecting the empty vector pEGFP-N1 or HBV encoding X gene expression,HBx protein in the cells was observed by immunofluorescence staining,and the mRNA and protein levels of interferon gene stimulator(STING)in cells were analyzed by real-time PCR and Western blot,respectively.The empty vector pEGFP-N1 and the HBV-encoding X gene expression plasmid pEGFP-HBx were transfected into HepG2 cells,respectively,and the mRNA and protein levels of STING and ring finger protein 5(RNF5)cells were analyzed.Subsequently,RNF5 was silenced with siRNA technology,and the expression levels of STING and interferonβ(IFN-β)proteins in cells were analyzed.The HepG2 cells transfected with pEGFP-HBx were then treated with different concentrations of protease inhibitor MG132,and the expression levels of STING and IFN-βprotein in HepG2 cells were analyzed.Results There were no significant difference in the relative gene levels of STING in HepG2 cells transfected with empty vector,pHBV-48 and pHBV-48-X0 respectively(P>0.05);however,the relative protein levels of STING in HepG2 cells transfected with empty vector(1.14±0.11)were higher than that in transfected pHBV-48 HepG2 cells(0.47±0.09)(P<0.01),but lower than those of HepG2 cells transfected with pHBV-48-X0(2.15±0.18)(P<0.01).In addition,the relative gene levels of STING gene in HepG2 cells transfected with empty vector and pEGFP-HBx had no statistical difference(P>0.05),but the relative protein levels of STING in HepG2 cells transfected with pEGFP-HBx(0.48±0.16)were lower than that in HepG2 cells transfected with empty vector(0.87±0.22).Compared with HepG2 cells transfected with empty vector,the relative gene and prot

关 键 词：乙型肝炎病毒 HBX RNF5 STING/IFN-β信号通路 

分 类 号：R373.21[医药卫生—病原生物学]