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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 To investigate the molecular mechanism of hepatitis B virus X protein regulating the promoter methylation of sFRP 1 and sFRP5 in order to guide the clinical treatment of diseases caused by hepatitis B virus infection. Methods The strain HBV was selected and cultured, and the DNA was extracted. After purification, methylation specific PCR and sulfide sequencing PCR were carried out respectively to sequence the target gene. Results HepG2.2.15 cells stably expressing HBV sFRP1, sFRP5 gene promoter CpG island methylation degree higher than HepG2 cells, DAC-stable HepG2.2.15 cells stably expressing HBV sFRP1, sFRP5 promoter The degree of methylation of CpG island in the region was partially decreased and dose dependent. Hepatitis B virus X protein can induce methylation of sFRP1 and sFRP5 promoter regions; DAC can reverse the degree of methylation of sFRP1 promoter region induced by HBV X protein in a dose-dependent manner; sFRP1 At gene amplification, the degree of methylation in the HBx group was higher than in the GFP group. Conclusion Hepatitis B virus x protein is enriched in the promoter region of sFRP1 gene and sFRP5 gene by inducing related methylated transferase and methylated CpG adhesion protein to promote methylation of gene CpG island, Can induce histone deacetylation, eventually leading to the down-regulation or silencing of sFRP1 and sFRP5 gene expression.