5-氮杂-2’-脱氧胞苷和曲古抑菌素A对胃癌细胞中P16和hMLH1及MGMT基因启动子甲基化和mRNA表达的影响  

Effects of 5-Aza-2＇-deoxycytidine and trichostatin A on P16, hMLH1 and MGMT genes and DNA methylation in human gastric cancer cells

作　　者：孟春风[1] 朱新江[1] 戴冬秋[1] 彭过[1] 

机构地区：[1]中国医科大学附属第一医院肿瘤外科普通外科教研室,沈阳110001

出　　处：《中华胃肠外科杂志》2009年第5期494-497,共4页Chinese Journal of Gastrointestinal Surgery

基　　金：基金项目：国家自然科学基金资助项目（30572162）;辽宁省教育厅资助项目（2008S240）

摘　　要：目的探讨DNA去甲基化制剂-5-氮杂-2’-脱氧胞苷（5-Aza-dC）和组蛋白去乙酰化酶抑制剂一曲古抑菌素A（TSA）对体外培养的胃癌细胞MGC-803中P16、hMLH1和MGMT基因启动子区DNA甲基化及基因表达的影响。方法应用5-Aza-dC和TSA处理体外培养的胃癌细胞MGC-803后。甲基化特异性聚合酶链反应法检测用药后细胞中P16、hMLH1和MGMT基因启动子区DNA甲基化状态；RT—PCR检测用药后细胞中P16、hMLH1和MGMTmRNA的表达水平。结果胃癌细胞系MGC-803中P16、hMLH1和MGMT基因mRNA表达缺失．其启动子区域表现为DNA高甲基化。5-Aza-dC不但能使P16、hMLH1和MGMT基因发生DNA去甲基化，而且能使表达缺失的P16、hMLH1和MGMTmRNA获得表达；TSA不能使上述基因发生去甲基化。亦不能使P16和hMLH1mRNA表达：5-Aza-dC和TSA联合作用下，上述3种基因的mRNA相对表达水平分别为0．412±0．030、0．397±0．024和0．553±0．043，明显高于5-Aza-dC单独作用（0．221±0．022、0．214±0．018和0．156±0．017，均P〈0．05）。结论启动子区DNA高甲基化是胃癌细胞中P16、hMLH1和MGMT基因失活的主要原因之一，5-Aza-dC单独应用及5-Aza-dC和TSA联合应用致胃癌细胞中P16、hMLH1和MGMT基因启动子去甲基化．从而使其重获表达。Objective To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-dC） and trichostatin A （TSA） on DNA methylation and expression of P16, hMLH1 and MGMT genes in the human gastric cancer cell line MGC-803, and to explore the mechanism of P16, hMLH1 and MGMT gene silencing in human gastric cancer cells. Methods MGC-803 cells were cultured in RPMI-1640 medium and were treated with 5-Aza-dC or TSA. Methylation-specific polymerase chain reaction （MSPCR） was used to detect the promoter methylation status of P16, hMLH1 and MGMT genes. RT-PCR was used to detect the mRNA expressions o.f P16, hMLH1 and MGMT. Results Promoter hypermethylation of P16, hMLH1 and MGMT genes were detected in MGC-803 cells, and mRNA expressions of P16, hMLH1 and MGMT were absent before treatment. After treatment with 5-Aza-dC, the promoter region of the P16, hMLH1 and MGMT gene exhibited a demethylation status, and their mRNA expressions were increased. The treatment with TSA had no effects on DNA demethylation or restoration of P16 or hMLH1 expression. P16, hMLH1 and MGMT mRNA relative expression levels after treatment with a combination of 5-Aza-dC and TSA were 0.412±0.030, 0.397±0.024 and 0.553±0.043 respectively, which were higher than those after 5-Aza-dC treatment alone（0.221±0.022, 0.214±0.018 and 0.156±0.017, all P〈0.05）. Conclusions Promoter hypermethylation is a major mechanism of P16, hMLH1 and MGMT gene silencing in human gastric cancer cells. Treatment with 5-Aza-dC alone or the combination of 5-Aza-dC and TSA can reactivate the expressions of these genes.

关 键 词：胃肿瘤 5-氮杂-2’-脱氧胞苷 曲古抑菌素A DNA甲基化 基因 P16 基因 HMLH1 基因 MGMT 

分 类 号：R735[医药卫生—肿瘤]