乙型肝炎病毒X蛋白通过降低P4启动子甲基化水平上调人IGF-Ⅱ基因的转录

HBx reduces methylation level of P4 promoter and up-regulates human IGF-Ⅱ gene transcription

目的:构建稳定表达乙型肝炎病毒X蛋白(HBx)的肝癌细胞株HepG2-HBx,探讨HBx对胰岛素样生长因子Ⅱ(IGF-Ⅱ)基因P4启动子甲基化水平及转录表达的影响。方法:应用基因重组技术,构建含HBx基因的重组逆转录病毒载体pBABE-puro-HBx,采用磷酸钙共沉淀法将其转染293FT包装细胞产生逆转录病毒,感染HepG2肝癌细胞,采用嘌呤霉素进行阳性克隆筛选,Western blotting鉴定表达HBx蛋白的肝癌细胞株HepG2-HBx。采用亚硫酸氢盐测序法及实时荧光定量RT-PCR检测HepG2-HBx细胞中P4启动子甲基化水平及P4mRNA表达水平变化。进一步将体外甲基化的人IGF-Ⅱ基因P4启动子驱动的荧光素酶报告载体pGL3-P4及含HBx基因的pCMV-tag2B-X质粒共转染HepG2肝癌细胞,采用亚硫酸氢盐测序法及双萤光素酶实验检测pGL3-P4载体上P4启动子甲基化水平及转录调控活性变化。结果:(1)经Western blotting鉴定,成功构建了稳定表达HBx蛋白的肝癌细胞株HepG2-HBx;(2)表达HBx蛋白的HepG2-HBx细胞中P4启动子甲基化CpG位点的比例(9.0%)明显低于对照细胞HepG2-control(25.0%)(P<0.01),而其P4 mRNA表达水平则为对照细胞HepG2-control的2.8倍;(3)共转染pCMV-tag2B-X质粒的HepG2细胞中pGL3-P4载体上P4启动子甲基化CpG位点的比例(60.8%)明显低于共转染对照质粒pCMV-tag2B的HepG2细胞(84.1%)(P<0.01),而前者P4启动子相对萤光素酶活性(14.12±0.89)则明显高于后者(4.61±0.76)(P<0.01)。结论:HBx蛋白可降低IGF-Ⅱ基因P4启动子甲基化水平,进而上调其转录表达。

AIM：To establish HepG2-HBx cell line stably expressing hepatitis B virus X protein（HBx） and to investigate the effect of HBx on the methylation status and transcription activity of human insulin-like growth factor II（IGF-II） gene P4 promoter. METHODS：HBx gene was cloned into the pBABE-puro retrovirus vector to construct recombinant plasmid pBABE-puro-HBx. The latter was transfected into 293FT package cells to generate retrovirus-pBABE-puro-HBx. HepG2 cells were infected with the virus suspension and the resistant cell clones were selected by puromycin. HBx expression in the HepG2-HBx cells was analyzed by Western blotting. P4 promoter methylation status and P4 mRNA expression in HepG2-HBx cells and HepG2-control cells were detected by bisulfite sequencing and real-time fluorescence quantitative RT-PCR, respectively. Furthermore, in vitro methylated pGL3-P4 vector driven by P4 promoter of human IGF-II gene was co-transfected into HepG2 cells with pCMV-tag2B-X plasmid carrying HBx gene or control empty plasmid pCMV-tag2B, and the methylation status and transcription activity of P4 promoter in pGL3-P4 vector were analyzed by bisulfite sequencing and dual-luciferase reporter assay system, respectively. RESULTS：HepG2-HBx cell line stably expressing HBx was successfully constructed. The percentage of methylated CpG dinucleotides in P4 promoter was lower in HepG2-HBx cells（9.0%） than that in HepG2-control cells（25.0%）（P〈0.01）, and P4 mRNA expression level of the former was 2.8 times higher than that of the latter. The percentage of methylated CpG dinucleotides in P4 promoter at pGL3-P4 vector was lower in the HepG2 cells co-transfected with pCMV-tag2B-X（60.8%） than that in the HepG2 cells co-transfected with control empty plasmid pCMV-tag2B（84.1%）（P〈0.01）, and the relative luciferase activity of the former（14.12？0.89） was higher than that of the latter（4.61？0.76）（P〈0.01）. CONCLUSION：HBx may reduce the methylation level of P4 promoter and up-regulates human IGF-II gene transcription.