胃癌形成过程中p16IN4a、Runx3和O-6-甲基鸟嘌呤-DNA甲基转移酶基因甲基化及其表达研究

目的 研究胃癌形成过程中p16INK4a、Runx3和O-6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因启动子区的高甲基化状态,同时检测MGMT的蛋白表达情况.探讨抑癌基因启动子区高甲基化与胃癌发生的关系.方法 选择经透明帽法进行首次黏膜病变切除者43例,其中异型增生27例,早期胃癌16例.选择胃镜活检证实为慢性萎缩性胃炎伴肠上皮化生者14例.另取20例正常胃黏膜活检组织作为对照.采用甲基化特异聚合酶链反应(MSP)检测每例组织中p16INK4a、Runx3和MGMT基因启动子区的甲基化状态,对所有甲基化p16INK4a产物进行测序,免疫组化检测MGMT蛋白表达情况.结果 肠上皮化生、异型增生和早期胃癌中p16INK4a基因甲基化率依次为14.3%(2/14)、22.2%(6/27)和37.5%(6/16);Runx3基因甲基化率依次为14.3%(2/14)、48.1%(13/27)和50.0%(8/16);MGMT基因甲基化率依次为7.1%(1/14)、48.1%(13/27)和50.0%(8/16).20名正常对照均未检出基因甲基化,与异型增生和早期胃癌相比差异有统计学意义(P＜0.05).Runx3和MGMT两种基因在异型增生和早期胃癌中的甲基化率显著高于肠上皮化生组(P＜0.05).各组病变中三种基因甲基化联合分析发现,异型增生和早期胃癌中甲基化的基因种类高于肠上皮化生组.差异有统计学意义(P＜0.01).基因甲基化与息者年龄、性别、幽门螺杆菌感染以及病变部位无相关性,但p16INK4a和MGMT基因甲基化与血清癌胚抗原水平升高显著相关(P值分别为0.003和0.039).MGMT基因启动子区高甲基化与其蛋白失表达密切相关(χ2=12.821,P=0.001).结论 抑癌基因启动子区高甲基化是基因失活的主要机制,可能是胃癌发生的早期分子事件.p16INK4a、Runx3和MGMT基因启动子区高甲基化在胃癌形成过程中起着重要的作用.

Study on methylation and expressions of p16INK4a,Runx3 and O-6-methylguanine-DNA methyltransferase genes in gastdc carcinogenesis

Objective To investigate the promoter methylation of p16INK4a,Runx3 and O-6-methylguanine-DNA methyltransferase genes(MGMT)and protein expression of MGMT in order to understand the relationship between high methylation and carcinogenesis.Methods Fourteen cases of chronic atrophic gastritis with intestinal metaplasia(IM).27 with dysplasia(DY)and 16 with early gastric cancer(EGC)were enrolled,and 20 normal gastric tissues served as controls.The methylation of p16INK4a,Runx3 and MGMT were measured by methylation-specific PCR,and the products of methylated p16INK4a gene were sequenced.The expression of MGMT protein was detected using immunohistochemistry.Results Methylation of p16INK4a was found in 14.3%(2/14)of IM,22.2% (6/27)of DY and 37.5%(6/16)of EGC.Methylation of Runx3 was found in 14.3%(2/14)of IM,48.1%(13/27)of DY and 50.0%(8/16)of EGC.Methylation of MGMT was found in 7.1%(1/14) of IM,48.1%(13/27)of DY and 50.0%(8/16)of EGC.No methylation was detected in normsl tissues.The methylation frequency of each gene in DY and EGC was significantly higher than that in normal tissues(P＜0.05).Furthermore,the methylation frequency of Runx3 and MGMT in DY and EGC was significantly higher than those in IM(P＜0.05).There was no significant difference in methylation frequency between DY and EGC.The number of methylated genes in DY and EGC was significantly higher than that in IM(P＜0.05).Promoter methylation of MGMT was strongly associated with loss of MGMT protein(P＜0.01).There was no significant correlation between methylation status and clinical pathological characteristics.but the promoter methylation of p16INK4a and MGMT was related with elevated CEA in serum(P＜0.01).Conclusions Promoter hypermethylation is the predominant mechanism of inactivation of tumour suppressor genes and may be an early event in the pathogenesis of gastric carcinoma.Promoter hypermethylation of p16INK4a,Runx3 and MGMT may be involved in the gastric carcinogenesis.