燃煤污染型砷中毒人群MGMT基因甲基化、转录及表达的研究

Hypermethylation and Transcription and Expression of O^6-methylguanine-DNA Methyltransferase Gene in Patients of Endemic Arsenism

目的了解O6-甲基鸟嘌呤DNA甲基转移酶基因(MGMT基因)启动子区甲基化、转录及表达以及DNA甲基转移酶1(DNMT1)的转录及表达与砷中毒的关系。方法采集68例燃煤污染型砷中毒(以下简称砷中毒)患者(轻度24例、中度28例、重度16例)及23例非病区居民外周血。用甲基化特异性PCR法(MSP)检测MGMT基因启动子区甲基化,实时荧光定量PCR(FQ-PCR)法检测MGMT及DNMT1 mRNA的水平。利用自愿手术治疗的砷中毒患者皮肤组织标本(61例,其中34例为一般病变,21例为癌前病变,6例为癌变)和对照皮肤组织标本(15例),以免疫组织化学(IHC)法检测砷中毒患者及对照皮肤组织中MGMT及DNMT1蛋白的表达。结果 MGMT基因启动子区甲基化阳性率与砷中毒程度有关(χ2=13.739,P<0.01)。砷中毒组MGMT mRNA表达水平与对照组比较,差异无统计学意义(P>0.05);MGMT基因启动子区甲基化患者和非甲基化患者之间MGMT mRNA和DNMT1 mRNA表达差异无统计学意义(P>0.05)。但轻、中度患者DNMT1 mRNA表达明显低于对照组(P<0.01);癌前病变组和癌变组皮肤组织中MGMT蛋白表达明显低于对照组(P<0.01),与皮肤损害程度有关(rs=-0.446,P<0.01);MGMT基因启动子区甲基化患者MGMT蛋白表达明显低于非甲基化组(P<0.05)。砷中毒组皮肤组织中DNMT1蛋白表达强于对照组(P<0.01),并与皮肤损害程度有关(rs=0.740,P<0.01);且MGMT基因启动子区甲基化患者组织中DNMT1蛋白表达明显高于非甲基化组(P<0.05)。结论 MGMT基因启动子区高甲基化抑制了燃煤污染型砷中毒患者皮肤组织中MGMT蛋白的表达,且DNMT1蛋白的高表达参与了此抑制过程。

Objective To investigate the promoter hypermethylation, transcription and expression of O^6-methylguanine-DNA methyltransferase gene （MGMT）, the transcription and expression of DNA methyhransferase 1 （DNMT1）, and the relationship between these changes and the arsenism. Methods The blood samples were collected from 68 arsenism patients （including 24 mild cases, 28 moderate cases and 16 severe cases）and 23 controls （non-arsenic exposure）. The mRNA expression of MGMT and DNMT1 was detected by real-time quantitative reverse transcription polymerase chain reaction, at the same time, the tissue samples of skin were collected from the patients with endemic arsenism （total 61 cases, including 34 general pathological changes cases, 21 precancerous cases and 6 cancerous cases） and from 15 controls. MGMT and DNMT 1 proteins were detected by immunohistochemical method. Results The positive rates of the promoter hypermethylation of MGMT were associated with the degree of arsenic poisoning （Х^2=13.739,P〈0.01）. The average level of MGMT mRNA showed no significant difference between arsenism patients and controls （P〉0.05）, and no significant difference of the average level of MGMT and DNMT1 mRNA was seen between methylation group and non-methylation group of the promoter of MGMT （P〉0.05）,but the average levels of DNMT1 mRNA of mild and moderate groups were significantly lower than that of the control group （P〈0.01）. Compared with the control group, the MGMT protein expression was lower in precancerous and cancerous groups （P〈0.01）,which was associated with the degree of skin damage（rs=-0.446, P〈0.01 ）; the MGMT protein expression in promoter methylation group of MGMT was lower than that in non-methylation group （P〈0.05）.Compared with the control group, the DNMT1 protein expression was higher in arsenism group （P〈0.01）, which was associated with the degree of skin damag （r,=0.740, P〈0.01）; the DNMT1 protein expression in promoter methylation group of MGMT was higher than that in non-methylation group （P〈0.05）. Conclusion The promoter hypermethylation of MGMT inhibits the protein expression of MGMT, and the high expression of DNMT1 protein participates in the inhibition process.