乙型肝炎病毒X蛋白通过诱导P3启动子低甲基化促进胰岛素样生长因子-II基因表达  被引量:2

Hepatitis B virus X protein promotes insulin-like growth factor H gene expression by inducing hypomethylation of the P3 promoter in hepatocellular carcinoma

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作  者:汤绍辉[1] 张少华[1] 张小娟[1] 吴胜兰[1] 李俊峰[1] 江向武[1] 周鸿科[1] 罗羽宏[1] 曹明溶[1] 

机构地区:[1]暨南大学附属第一医院消化科,广州510630

出  处:《中华肝脏病杂志》2014年第4期289-294,共6页Chinese Journal of Hepatology

基  金:广东省自然科学基金(9151008901000001)I广东省医学科学技术研究基金(A2009367,A2012332);中央高校基本科研业务费专项资金(21610407)暨南大学附属第一医院科研培育基金(2012109)

摘  要:目的探讨乙型肝炎病毒X蛋白(HBx)对胰岛素样生长因子-II(IGF—II)基因P3启动子驱动的mRNA表达的影响及其表观遗传机制。方法分别采用定量RT-PCR和亚硫酸氢盐测序法检测HBV感染阳性和阴性肝癌组织中P3mRNA、HBxmRNA表达水平及P3启动子甲基化状态;进一步分析稳定表达HBx蛋白的肝癌细胞株HepG2-HBx及其对照细胞株HepG2-control中P3mRNA表达水平及P3启动子甲基化状态;最后,将体外甲基化的IGF—II基因P3启动子驱动的荧光素酶报告载体pGL3-P3及携带HBx基因的pCMV-tag2B—X质粒共转染HepG2细胞,采用亚硫酸氢盐测序法及双萤光素酶试验检测HBx蛋白对P3启动子转录活性和甲基化状态的影响。计数资料采用x^2或Fisher精确检验,计量资料采用独立样本f检验或Mann-WhitneyU检验,线性相关分析采用Pearson相关系数评价。结果(1)HBV感染阳性的肝癌组织中P3mRNA表达水平(△cT为-9.59±3.22)明显高于HBV阴性的肝癌组织(△cT为-12.97±3.08),P=0.006;而P3启动子17个cpG位点的平均甲基化水平(36.9%土15.5%)明显低于HBV阴性的肝癌组织(52.1%±19.1%),P:0.025;P3mRNA丰度与HBxmRNA表达水平呈正相关,而与P3启动子甲基化水平呈负相关。(2)在HepG2-HBx及其对照细胞中,也显示了类似的P3mRNA表达水平及P3启动子甲基化水平变化。(3)体外转染的HBx蛋白显著降低了P3启动子甲基化水平,提高了其转录活性。结论HBx蛋白可能通过降低P3启动子甲基化水平促进肝细胞癌IGF—II基因表达,本结果为充分理解HBx蛋白介导的肝细胞癌发病机制提供了新的信息。Objective To explore the involvement of hepatitis B X protein (HBx) in promoter 3 (P3)-driven mRNA overexpression of the insulin-like growth factor II gene (IGF-II) and investigate the underlying epigenetic mechanism. Methods Levels of P3 and HBx mRNA and status of P3 methylation were analyzed in human hepatocellular carcinoma (HCC) samples, with and without hepatitis B virus (HBV) infection, using quantitative reverse transcription-PCR and bisulfite sequencing. In addition, the levels of P3 mRNA and P3 methylation were examined in HepG2 cells stably overexpressing HBx (HepG2-HBx). Finally, P3 promoter-luciferase constructs were cotransfected into HepG2 cells along with an HBx-expressing plasmid, and the effects of HBx on transcriptional activity and methylation of P3 were analyzed. Statistical analyses of the data were conducted by chi square test, Fisher's exact test, Student's t-test, Mann-Whitney U test, and Pearson's correlation coefficient test. Results The HBV-positive HCC specimens had significantly higher levels of P3 mRNA than the HBV-negative HCC specimens (-9.59 ± 3.22 vs. -12.97 ± 3.08△ CT;P = 0.006) but significantly lower levels of P3 methylation (mean values for the 17 CpG sites (36.9% ± 15.5% vs. 52.1% ± 19.1%;P = 0.025). The P3 transcript abundance was positively correlated with the level of HBx expression and negatively correlated with the level of P3 methylation. The epigenetic results from experiments with the HepG2-HBx cells were similar. Transfection of HBx significantly decreased P3 methylation level and increased its activity. Conclusion HBx expression may promote IGF-II expression by inducing hypomethylation of its P3 promoter in hepatocellular carcinoma.

关 键 词: 肝细胞 X蛋白 肝炎病毒 乙型 胰岛素样生长因子II 启动子 低甲基化 

分 类 号:R512.62[医药卫生—内科学]

 

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