乙型肝炎病毒复制对肝癌细胞Arid2基因表达的影响  

Replication of hepatitis B virus induces down-regulation of Arid2 expression in hepatoma cells

作　　者：李治[1] 陈震[1] 段玉洁[1] 聂丽珠[1] 田玲[1] 唐霓[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2015年第3期344-349,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81371827;31171307);"十二.五"传染病国家科技重大专项资助(编号:2012ZX10002005-003-002;2013ZX10002002-005-003);重庆医科大学附属第二医院"优秀青年人才"资助项目

摘　　要：目的:初步研究乙型肝炎病毒(hepatitis B virus,HBV)感染及其复制对肝癌细胞中抑癌基因Arid2(AT-rich interactive domain 2)表达的影响。方法:采用携带HBV基因组1.1倍体的复制型重组腺病毒Ad-HBV1.1感染Huh7细胞,或者四环素诱导的HBV复制细胞模型Hep AD38细胞,观察HBV瞬时或稳定复制细胞模型中Arid2的表达变化;进一步采用HBV各元件乙型肝炎病毒S蛋白(hepatitis B virus S protein,HBs)、乙型肝炎病毒DNA聚合酶(HBV-pol)、乙型肝炎病毒核心蛋白(hepatitis B virus core protein,HBc)、乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)的过表达质粒转染Huh7细胞,明确病毒自身编码蛋白对Arid2表达的调控作用。在上述细胞模型中,运用Southern-blot、ELISA、real-time PCR的方法验证HBV的感染与复制情况,通过RT-PCR、Western blot检测肝癌细胞抑癌基因Arid2表达水平的变化。结果:HBV瞬时转染或稳定复制细胞模型中,Ad-HBV1.1感染的Huh7细胞m RNA和蛋白表达水平明显降低,Western blot结果显示Arid2蛋白表达水平降低了46.10%(P<0.05);在HBV稳定复制的Hep AD38细胞中,Arid2蛋白表达水平降低了37.63%(P<0.05)。转染HBV 4种病毒编码蛋白过表达质粒的Huh7细胞中,过表达HBs、HBx组较其他实验组Arid2表达水平明显降低,Western blot结果显示Arid2蛋白表达水平在HBs组降低了54.55%(P<0.05),在HBx组降低了46.38%(P<0.05)。结论:HBV的感染和复制导致肝癌细胞中Arid2的表达水平下调,Arid2基因的表达与HBV复制水平呈负相关,其中HBs、HBx对Arid2的下调作用较明显。Objective：To investigate the effects of hepatitis B virus replication on the expression of Arid2 in hepatoma cells. Methods：The HBV-stable or transient expression cell models were used to evaluate the regulatory effects of HBV replication on Arid2 expression. For stable replication model,Hep AD38 cells were used to express HBV under the control of inducible tetracycline promoter. For transient expression system,Huh7 cells were infected with the replicative recombinant adenovirus Ad-HBV1.1 carrying 1.1 copies of HBV genome. Southern-blot,ELISA and real-time quantitative PCR were employed to confirm HBV replication efficiency in hepatoma cells. The expression levels of Arid2 gene were determined by RT-PCR and Western blot. To further identify which of the viral-encoded proteins implicated in the regulation of Arid2 expression,Huh7 cells were transfected with hepatitis B virus S protein（HBs）,HBp,hepatitis B virus core protein（HBc）or hepatitis B virus X protein（HBx）-expression plasmids,and Arid2 expression level were determined. Results：HBV viroloigal marker and replicative intermediates were detected both in HBV-stably or transient expression system,indicating that HBV-stably or transiently expression cell model can robustly support HBV replication. The expression levels of Arid2 gene were significantly down-regulated both in the HBV stable replication and transient replication cell model. The protein expression level of Arid2 was decreased by 46.10% in Huh7 cells transfected with Ad-HBV 1.1 and by 37.63% in Hep AD38 cells（P0.05）. Among the four viral-encoded expression plasmids,HBV surface antigen and HBx significantly repressed Arid2 m RNA and protein expression,with inhibitory effects of 54.55%and 46.38%（P0.05）in protein level by HBs and HBx respectively. Conclusion：HBV replication represses Arid2 expression in hepatoma cells. Among viral-encoded proteins,HBs and HBx may play important roles in HBV-induced Arid2 repression.

关 键 词：乙型肝炎病毒 Arid2 乙型肝炎病毒S蛋白 乙型肝炎病毒X蛋白 

分 类 号：R512.62[医药卫生—内科学]