应用shRNA研究乙型肝炎病毒X基因表达和三氧化二砷对HepG2细胞p53表达和活性的影响  

Study of effects of HBV X gene and As_2O_3 on expression and activity of p53 in HepG2 cells with shRNA

作　　者：贺兴鄂[1] 雷建华[1] 杨旭[1] 王文龙[1] 罗红雨[1] 梁骏[1] 

机构地区：[1]中南大学湘雅二医院肝病研究中心,长沙410011

出　　处：《中华肝脏病杂志》2006年第10期757-761,共5页Chinese Journal of Hepatology

基　　金：国家自然科学基金(30371402)

摘　　要：目的应用短发夹状RNA介导的RNA干扰研究乙型肝炎病毒X基凶(HBX)和三氧化二砷(As_2O_3)对HepG2细胞p53表达和活性的影响。方法以2μmol/L As_2O_3处理HepG2细胞和表达HBX的HepG2-X,以未处理细胞为对照,提取细胞裂解物或胞核袋解物,采用改进的酶联免疫吸附法检测p53总量和相对活性吸光度。应用脂质体介导具钉HBX序列特异性短发央状RNA表达载体Xi S1、Xi-S2和序列无关对照Xi-S3转染HepG2 X,再次观察As_2O_3处理对p53表达和活性的影响。结果As_2O_0_3作用使HepG2和HepG2 X细胞p53蛋白水平上调和p53相对活性比增强。表达HBX后As_2O_3秀导的p53蛋白水平有所上调,但显著抑制p53相对活性。短发夹状RNA抑制HBX的表达后其对p53的活性抑制得以解除。结论As_2O_3使HepG2细胞p53表达上调和活性增强。应用短发夹状RNA介导的RNA干扰有利于研究HBX的表达对p53表达和活性的影响。HBX表达上调As_2O_3诱导的p53蛋白水平,但显著抑制p53的结合与转录调节活性。Objective To delineate the effects of HBV X gene and of As203 on p53 expression and activity in HepG2 cells by shRNA-mediated RNA interference （RNAi）. Methods HepG2 cells and cells with stable expression of HBV X gene, HepG2-X, were treated with 2 μmol/L As2O3, and the corresponding untreated cells were used as controls, Cell and nuclear lysates were extracted, Total level and the relative activity absorbance of p53 were detected by modified ELISA, HBV X gene sequence-specific shRNA expression vectors, Xi-S 1 and Xi-S2, and sequence-unrelated control Xi-S3 were transfected into HepG2-X. The effect of As：O3 on p53 expression and activity were retested, Results Total p53 level was up-regulated and its relative activity ratio was enhanced by As2O3 in HepG2 and HepG2-X cells. The total p53 level induced by As2O3 was further upregulated by HBX expression, while its relative activity was significantly suppressed. The suppression was removed after HBX expression was suppressed by shRNA. Conclusion As2O3 could up-regulate p53 expression and enhance its activity, shRNA- mediated RNA interference is conveniently being used in studies on the effect of HBV X gene expression on p53 expression and activity. I-IBV X expression could up-regulate p53 gene expression level induced by As2O3, while it suppressed the activity of p53.

关 键 词：肝炎病毒 乙型 RNA干扰 P53蛋白质 三氧化二砷 基因 X 

分 类 号：R735.7[医药卫生—肿瘤]