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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 investigate the regulatory mechanism of hepatitis B virus X protein (HBx) involved in hsp90α gene expression in human hepatocellular carcinoma cells. Methods: The expression of hsp90α gene and its promoter activity in HepG2 cells transfected with different doses of HBx expression vector were detected by semi-quantitative RT-PCR, Western blotting and luciferase assay. The transcription factor c Effect of Myc blocker 10058-F4 on these effects. The promoters were used to analyze the activation of hsp90α gene promoter by c-Myc. The expression of c-Myc mRNA and protein in HepG2 cells expressing HBx was detected by RT-PCR and Western blotting. Western blot and ELISA were used to detect the expression of ERK1 / 2 and phosphorylated ERK1 / 2 -ERK1 / 2) expression and endogenous ERK activity. Results: The mRNA and protein expression of hsp90α gene and the promoter activity of HepG2 cells were increased in a dose-dependent manner (P = 0.006 1). The c-Myc inhibitor inhibited the up-regulation of hsp90α gene expression by HBx gene. c-Myc activates the hsp90α gene promoter activity by binding to the c-Myc site (-1094 / -1088). HBx enhanced the expression of c-Myc mRNA and protein in HepG2 cells, accompanied with an increase of phosphorylation of ERK1 / 2 protein and a significant increase of ERK activity (P = 0.032). CONCLUSION: HBx transactivates c-Myc-mediated promoter activity of hsp90α gene in hepatocellular carcinoma cells through ERK signaling pathway, thereby up-regulating hsp90α gene expression.