乙型肝炎病毒X蛋白调控sFRP1和sFRP5启动子区的甲基化修饰及其分子机制的探讨

Examination of HBV X protein,which regulates the methylation of the sFRP 1 and sFRP5 promoter regions,and its molecular mechanisms

目的探讨乙型肝炎病毒X蛋白调控sFRP 1、sFRP5启动子区的甲基化修饰分子机制,以期指导乙型肝炎病毒感染所致疾病的临床治疗。方法选取标本株HBV并对其进行培养,提取DNA,纯化后分别进行甲基化特异性PCR和硫化测序PCR,对目的基因进行测序。结果稳定表达HBV的HepG2.2.15细胞株sFRP1、sFRP5基因的启动子区CpG岛的甲基化程度比HepG2细胞株高,经DAC处理的稳定表达HBV的HepG2.2.15细胞株的sFRP1、sFRP5启动子区CpG岛甲基化程度有部分回落且呈药物剂量依赖。乙型肝炎病毒X蛋白可对sFRP1、sFRP5基因的启动子区的甲基化起到诱导作用;DAC可逆转HBV X蛋白所诱导的sFRP1启动子区甲基化程度,且呈剂量依赖;进行sFRP1基因扩增时,HBx组的甲基化程度比GFP组高。结论乙型肝炎病毒x蛋白通过诱导相关的甲基化转移酶以及甲基化CpG粘附蛋白而富集于sFRP1基因和sFRP5基因的启动子区,从而促进基因CpG岛的甲基化,并且还可以诱导组蛋白的脱乙酰化,最终导致sFRP1基因和sFRP5基因的表达下调或沉默。

Objective The study investigated the molecular mechanisms by which the X protein of the hepatitis B virus regulates the methylation of the sFRP I and sFRP5 promoter regions in order to guide the clinical treatment of hepatitis B viral infections. Methods Samples of HBV strains were selected and cultured. DNA was then extracted and samples were purified. Afterwards, methylation-specific PCR and bisulfite sequencing PCR were performed, and the target genes were sequenced. Results The promoter region CpG island of the sFRP1 and sFRP5 genes in a HepG2.2.15 cell line that stably expressed HBV had a greater degree of methylation than in a HepG2 cell line. The promoter region CpG island of the sFRP1 and sFRP5 genes in the HepG2.2.15 cell line that stably expressed HBV bad a degree of methylation that decreased after DAC in a dose-dependent manner. The HBV X protein induced the methylation of the promoter region in the sFRP1 and sFRP5 genes. The degree of methylation of the promoter region induced by HBV X protein in the sFRP1 gene was reversed by DAC in a dose-dependent manner. When the sFRP1 gene was amplified, the degree of rnethylation was greater for HBx than for GFP, Conclusion Methyltransferase enzymes and methylated CpG adhesion proteins induced more abundant HBV X protein in the promoter region in the sFRP1 gene and sFRP5 gene, thereby promoting the methylation of the gene CpG island and also inducing histone deacetylation. This ultimately resulted in downregulation or silencing of the sFRP1 gene and sFRP5 gene.