人肝癌细胞株HepG2中原钙粘附蛋白10基因异常甲基化及其与HBx蛋白的关系  

Aberrant hypermethylation of protocadherin10gene and relationship with HBx protein in HepG2hepatoma carcinoma cells

作　　者：范颂[1] 黄世峰[1] 曹炬[1] 张莉萍[1] 

机构地区：[1]重庆医科大学附属第一医院检验科,重庆400016

出　　处：《临床检验杂志》2012年第5期371-374,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(30973378;81071621);重庆市自然科学基金(CSTC;2009BB5257);重庆医科大学附属第一医院院内医学科学基金(YXJJ2009-12)

摘　　要：目的研究原钙粘附蛋白10(PCDH10)启动子在人肝癌细胞株HepG2中的甲基化状态及与HBx调控的相关性。方法用逆转录PCR(RT-PCR)和甲基化特异性聚合酶链反应(MSP),检测去甲基化药物5-氮-2'-脱氧胞苷(Aza)联合组蛋白乙酰化酶抑制剂TSA(trichostatin A)处理前后,HepG2细胞中PCDH10基因的表达及启动子甲基化水平;用亚硫酸氢盐测序PCR(BSP)定位检测发生甲基化的CG位点。用MSP检测表达HBx基因的重组腺病毒感染HepG2细胞后PCDH10基因的表达及甲基化水平。结果 PCDH10基因在HepG2细胞株中表达沉默,其启动子发生明显甲基化,Aza可以部分去除PCDH10甲基化从而上调其mRNA表达水平。但HBx蛋白表达不影响HepG2细胞中PCDH10的甲基化程度。结论 PCDH10启动子异常甲基化使HepG2细胞内转录沉默,但其甲基化状态与HBx蛋白表达无关。Objective To investigate the methylation status of anti-oncogene protocadherin10 promoter in hepatoma cell line HepG2 and the relationship with the regulation of HBV X protein（HBx）.Methods The expressions of PCDH10 gene in HepG2 cells before and after the treatment with demethylating agent 5-aza-2′-deoxycytidine combined histone deacetylase inhibitors trichostatin A were detected by using reverse transcription PCR（RT-PCR）.The methylation levels of PCDH10 promoter CGI（CpG island） were detected by methylation-specific PCR（MSP）.The methylated CG sites were specifically detected by bisulphite sequencing PCR（BSP）.The transcriptional levels and the methylation status of PCDH10 in HepG2 cells treated by recombinant adenoviruses expressing HBx protein were also detected by MSP.Results PCDH10 gene was silenced in HepG2 cells and aberrant hypermethylation of PCDH10 promoter CGI was detectable in HepG2 cells.The transcriptional silence of PCDH10 could be partly reversed by Aza with pharmacologic demethylation and the expression of PCDH10 mRNA was up-regulated.No influences on hypermethylation of PCDH10 in HepG2 cells by the expression of HBx protein was found.Conclusion Aberrant hypermethylation of PCDH10 promoter CGI may result in transcriptional silence in HepG2 cells.However,no correlation was found between the expression of HBx protein and aberrant methylation of PCDH10.

关 键 词：肝细胞肝癌 甲基化 原钙粘附蛋白10 HBX蛋白 

分 类 号：R394.3[医药卫生—医学遗传学]