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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。
Objective: To construct a HepG2-HBx hepatocellular carcinoma cell line stably expressing Hepatitis B virus X protein (HBx) and investigate the effect of HBx on the promoter methylation and transcription of P4 gene of insulin-like growth factor Ⅱ (IGF-Ⅱ) gene. Methods: Recombinant retrovirus vector pBABE-puro-HBx containing HBx gene was constructed by gene recombination technique. The recombinant retrovirus vector pBABE-puro-HBx was transfected into 293FT packaging cells by calcium phosphate precipitation method to produce retrovirus and infected with HepG2 hepatoma cells. Puromycin Positive clones were screened and identified by Western blotting. HepG2-HBx cells expressing HBx protein were identified. The bisulfite sequencing and real-time fluorescent quantitative RT-PCR were used to detect the P4 promoter methylation level and P4 mRNA expression level in HepG2-HBx cells. Furthermore, the luciferase reporter vector pGL3-P4 driven by P4 gene promoter of human IGF-Ⅱ gene in vitro and the pCMV-tag2B-X plasmid containing HBx gene were co-transfected into HepG2 hepatoma cells. The bisulfite sequencing Luciferase assay and double luciferase assay were used to detect the P4 promoter methylation level and transcriptional regulatory activity in pGL3-P4 vector. Results: (1) The HepG2-HBx hepatocellular carcinoma cell line stably expressing HBx protein was successfully constructed by Western blotting. (2) The proportion of CpG locus of P4 promoter in HepG2-HBx cells expressing HBx protein (9.0 %) Was significantly lower than control HepG2-control (25.0%) (P <0.01), while P4 mRNA expression level was 2.8 times that of control HepG2-control; (3) cotransfection of pCMV-tag2B-X The proportion of CpG locus of P4 promoter (60.8%) in pGL3-P4 vector was significantly lower in HepG2 cells than in pCMV-tag2B (84.1%) (P <0.01) The relative luciferase activity of promoter (14.12 ± 0.89) was significantly higher than that of the latter (4.61 ± 0.76) (P <0.01). Conclusion: The HBx protein can reduce the promoter methylation level of IGF-Ⅱ gene and up-regulate its transcriptional expression.