乙型肝炎病毒X蛋白对人肝癌细胞hsp90α基因表达调控机制研究  

Regulation mechanism exploration of hsp90α expression by hepatitis B virus X protein in human hepatocarcinoma cells

作　　者：李卫华[1] 陈广原[1] 余湘文[1] 石永英[1] 彭妙官[1] 李慧[1] 卫建筠[1] 

机构地区：[1]广州医学院第一附属医院感染科,广东广州510102

出　　处：《中华肿瘤防治杂志》2013年第20期1574-1579,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：广东省自然科学基金(9151040701000037);广州市科技计划(2009J1-C321-2)

摘　　要：目的:探讨乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)参与人肝癌细胞hsp90α基因表达的调控机制。方法:采用半定量RT-PCR、蛋白质印迹法及荧光素酶活性检测技术,检测不同剂量HBx表达载体转染的HepG2细胞中hsp90α基因mRNA和蛋白表达及启动子活性的变化,并观察转录因子c-Myc阻断剂10058-F4对上述效应的影响。采用启动子实验分析c-Myc对hsp90α基因启动子的激活作用。RT-PCR和蛋白质印迹法检测表达HBx的HepG2细胞中c-Myc mRNA和蛋白表达情况;蛋白质印迹法及ELISA分析细胞外信号调节激酶1/2(ERK1/2)和磷酸化ERK1/2(p-ERK1/2)表达水平及内源性ERK的活性。结果:HBx基因转染HepG2细胞中hsp90α基因的mRNA和蛋白表达水平以及启动子活性呈剂量-依赖性增强,P=0.006 1;c-Myc阻断剂抑制了HBx对hsp90α基因表达的上调效应。c-Myc通过与c-Myc位点(-1094/-1088)结合转录激活hsp90α基因启动子活性。HBx使HepG2细胞中c-Myc mRNA和蛋白表达增强,并伴有ERK1/2蛋白磷酸化水平增加,ERK活性明显提高,P=0.032。结论:HBx通过ERK信号通路反式激活肝癌细胞c-Myc介导的hsp90α基因启动子活性,从而上调hsp90α基因表达。OBJECTIVE：To explore Hepatitis B virus X protein （HBx） in participanting the regulatory mechanism of heat shock protein 90 alpha （hspg0a） gene expression in human hepatoma cells. METHODS The expression of hspg0a mRNA and protein were analyzed by semi quantitative RT PCR and Western blot, and the transcriptional activity of hsp90a promoter was detected by luciferase assay in different doses of HBx-expressing vector transfected HepG2. The im pact of transcription factor c-Myc specific blocker, 10058-F4 on the effect was evaluated. The role of c-Myc in hsp90apro- moter activation was analyzed by promoter assay in HepG2 cells. The expression of c-Myc mRNA and protein were tested by RT-PCR and Western blot,and the expression of extracellular signal-regulated kinase 1/2（ERK1/2） and phosphoryla ted ERK1/2 （p-ERK1/2） were analyzed by Western blot, the endogenous ERKs activity was determined by ELISA assay in HepG2 expressing HBx. RESULTS： The expression of hspg0mRNA and protein and the hsp90a promoter activity were significandy increased in a dose dependent manner in HBx gene transfected HepG2 （P =0. 006 1）. The inductive effect of HBx was inhibited by c-Myc inhibitor in HepG2 expressing HBx. The c Myc transcriptional activated the hsp90a promoter activity through binding with c-Myc site（-1094/ 1088）. The expression of c Myc mRNA and protein as well as the phosphorylation of ERK1/2 were enhanced, ERKs activity was also improved in HepG2 expressing HBx （P=0. 032）.

关 键 词：人肝癌细胞株 乙型肝炎病毒X蛋白 hsp90α基因 转录因子c—Myc 表达调控 

分 类 号：R735.7[医药卫生—肿瘤]