谷胱甘肽硫转移酶P1基因DNA甲基化、mRNA和蛋白表达与燃煤污染型地方性砷中毒关系探讨

Relationship of DNA methylation, mRNA transcription and protein expression of glutathione-S-transferases- P1 gene and coal-pollution-borne endemic arsenism

目的探讨谷胱甘肽硫转移酶P1（GSTPl）基因启动子区DNA甲基化、mRNA和蛋白表达与砷中毒的关系。方法123例砷中毒患者来自贵州省兴仁县交乐村燃煤污染型地方性砷中毒病区，按地方性砷中毒诊断标准（WS／T211-2001），其中轻度42例、中度41例，重度40例。在距病区约13km的非砷暴露村．选择47例正常健康人作为对照。根据知情同意的原则，采集被调查者的外周血，用PCR法检测外周血GSTPl基因启动子区DNA甲基化，用荧光实时定量PCR法检测GSTPlmRNA表达。取自愿手术治疗的53例砷中毒患者的皮肤病理标本，其中一般病变28例、癌前病变20例、癌变5例，以病理学诊断无异常的15例非肿瘤手术患者的皮肤组织为对照组。采用免疫组织化学（IHC）法检测皮肤组织中GSTPl蛋白的表达。结果在按病情分组中，外周血GSTPl基因DNA甲基化阳性率轻（28．57％，12／42）、中（57．10％，23／41）、重度（65．00％。26／40）砷中毒组与对照组（6．38％，3／47）比较差异有统计学意义（x。值分别为7．792、26．000、33．412，P均〈0．01）；在按皮肤病理诊断分组中，GSTPl基因DNA甲基化阳性率一般病变（21．43％，6／28）、癌前病变（50．00％，10／20）、癌变组（80．00％，4／5）与对照组（6．67％，1／15）比较差异有统计学意义（x。值分别为3．562、7．468、10．756，P均〈O．05）。GsTPl基因DNA甲基化阳性率随砷中毒患者病情及皮肤病理损害程度加重而升高（趋势x。=38．239、x。=13．659，P均〈0．01）。与对照组（0．18426）比较，中（0．08777）、重度（0．05693）砷中毒组外周血GSTPlmRNA表达显著降低（P均〈0．01），其中重度砷中毒组低于中度砷中毒组（P〈0．01）；与对照组（0．33845）、一般病变组（0．27674）比较，癌前病变组（0．10481）、癌变组（0．04370）GSTPlmRNA表达显著降低（P均〈0．01），其中癌变组显著低于癌前病变组（P〈0．01）。皮肤组织中GSTPl蛋白阳性表达率组间比较差异有统计学意义（x2=20．948，P〈0．05），其中癌前病变组（65．00％，13／20）、癌变组（40．00％，2／5）与对照组（100．00％，15／15）比较差异有统计学意义（x2=12．183、11．778，P均〈0．01）。皮肤病损程度与GSTPl蛋白表达强度经Spearman等级相关分析，呈负相关关系（r=-0．520，P〈0．05）。按GSTPl基因DNA甲基化分组，GSTPlmRNA和蛋白表达阳性率与非甲基化组（0．18707、95．74％）比较，甲基化组（0．03840、57．14％）显著降低（Z=9．032，P〈0．01；x2=23．134，P〈0．01）。结论砷中毒可通过导致人体GSTPl基因启动子区DNA甲基化，进而抑制其mRNA和蛋白表达，GSTPl基因在砷致病或致癌过程中起重要作用。

Objective To investigate DNA methylation in the promoter region, mRNA transcription and protein expression of glutathione-S-transferases-P1 （GSTPI） gene and their relation with arsenism. Methods In endemic coal-pollution-borne arsenism area, Jiaole village of Xinren county, Guizhou province, according to the diagnostic criteria of endemic arsenism（WS/T 211-2001 ）, 123 cases with endemic arsenism were selected and divided into three groups （mild arsenism group： 42 cases, moderate arsenism group： 41 cases and severe arsenism group：40 cases）. Forty seven residents were selected as controls in a village about 12 km away from the endemic arsenism area. With the informed consent principle, peripheral blood of all respondents was collected in order to analyze DNA methylation and check mRNA. DNA methylation of GSTP1 gene promoter region in peripheral blood was assayed by PCR, and GSTP1 mRNA expression was assayed using real-time quantitative PCR. In addition, other cutaneous specimens originated from 53 cases with arsenism that accepted surgical treatment voluntarily were taken. Of these specimens, general pathological changes were 28 cases, precancerous 20 cases and cancerous 5 cases. Skin tissues of 15 cases of non-tumor surgery patients without abnormal pathological changes were as control group. GSTP1 protein expression in the skin tissue was detected using immunohistochemistry （IHC）. Results Among different groups of arsenic poisoning, the positive rate of DNA methylation of GSTP1 gene was 28.57% （12/42） in the mild group, 57.10%（23/41） in the moderate group and 65.00%（26/40） in the severe group. Compared with the control group （6.38%, 3/47 ）, the difference was statistically significant （~2 = 7.792, 26.000, 33.412, all P 〈 0.01）. Among different groups of arsenic poisoning diagnosed by dermapathology, the positive rate of DNA methylation of GSTP1 gene was 21.43%（6/28） in the general pathological change group, 50.00%（10/20） in the precancerous group and 80.00% （4/5） in the cancerous group. Compared with the control group （6.67%, 1/15）, the difference was statistically significant（x2 = 3.562, 7.468, 10.756, all P 〈 0.05）. It showed that the positive rate of DNA methylation of GSTP1 gene increased with aggravation of the disease and dermatic lesion of arsenism （tendency x2 = 38.239, x2 = 13.659, all P 〈 0.01 ）. Compared with the control group （0.184 26）, the expressions of GSTP1 mRNA in peripheral blood in moderate （0.087 77） and severe arsenic poisoning groups（0.056 93）were significantly reduced（all P〈 0.01 ）, and that of severe group was significantly lower than that of the moderate group（P 〈 0.01 ） ; compared with the control group （0.338 45） and the general lesion group （0.276 74）, GSTP1 mRNA expression was significantly reduced in precancerous lesion group（0.104 81） and cancerous group（0.043 70）, in which the cancerous group was significantly lower than that of the precancerous lesions. The difference of skin tissue GSTP1 protein expression rate between groups was statistically significant （~2 = 20.948, P 〈 0.05 ）, in which the difference between the precancerous lesion group（65.00%, 13/20）, the cancer group （40.00%, 2/5） and the control group （100.00%, 15/15） was statistically significant （X2= 12.183, 11.778, P 〈 0.01）. Spearman correlation analysis showed that the degree of skin lesion and the level of GSTP1 protein expression was negatively correlated （r = - 0.520, P 〈 0.05）. Groups Were divided according to DNA methylation of GSTP1 gene, and the mRNA and protein expression of GSTP1 in methylation group （0.038 40, 57.14%） was significantly lower compared with that of unmethylated group （0.187 07, 95.74% ; Z = 9.032, X2 = 23.134, all P 〈 0.01 ）. Conclusions Arsenism may lead to DNA methylation of human GSTP1 gene promoter region, thereby inhibiting expression of mRNA and protein. GSTP1 gene plays an important role in arsenism or carcinogenic process.