结肠癌组织RASSF1A基因转录表达和启动子区甲基化研究

Transcription,Expression and Promotor Hypermethylation of RASSF1A Gene in Human Colon Carcinoma Tissue

目的:研究Ras相关区域家族1A基因(ras association domain family 1A,RASSF1A)启动子区甲基化对结肠癌组织中该基因转录和表达的影响。方法:应用甲基化特异性PCR(Methylation-special PCR,MSP)、RT-PCR和Western blot方法检测30例结肠癌组织和癌旁组织中的RASSF1A基因启动子区甲基化状态、mRNA和蛋白表达水平。结果:①RASSF1A基因启动子区在结肠癌组织和正常组织中的甲基化频率分别为57%(17/30)和20%(6/30),甲基化频率在两组具有统计学差异(p<0.01),,结肠癌组织中RASSF1A基因启动子区甲基化频率显著高于癌旁正常组织(x2=8.531,p<0.01);②结肠癌组织中RASSF1A基因mRNA和蛋白表达均显著低于癌旁组织(癌组织和癌旁正常组织中mRNA相对表达量分别为0.2836±0.0493和0.5092±0.0433,P<0.001;以上组织中蛋白相对表达量分别为0.3124±0.0472和0.5320±0.0440,P<0.01);③在结肠癌组织中,甲基化组RASSF1A基因mRNA和蛋白表达明显低于非甲基化组(甲基化组和非甲基化组mRNA相对表达量分别为0.0686±0.0174和0.5511±0.0486,P<0.0001;以上组中蛋白相对表达量分别为0.1219±0.0326和0.5614±0.0380,P<0.0001)。结论:结肠癌组织中RASSF1A基因启动子区甲基化明显增高,与该基因蛋白表达减少显著相关,这可能是导致结肠癌中RASSF1A抑癌基因失活的主要原因。

Objective：To investigate the state of promotor methylation of RASSF1A gene in tissue of human colon carcinoma and analyze the relationship between the state of promotor methylation of RASSF1A gene and gene transcription and expression.Methods：Promotor methylation,mRNA and RASSF1A protein level were determined by Methylation-special PCR,RT-PCR and Western blot in cancer tissue and adjacent normal tissue from 30 colon carcinoma patients respectively.Results：① Methylation frequency of RASSF1A gene in cancer tissue was higher than that in adjacent normal tissue（57% VS 17%,P0.001）;mRNA and protein expressed by RASSF1A gene in cancer tissue were significantly less than those in adjacent normal tissue（mRNA：0.2836±0.0493 VS 0.5092±0.0433,P0.001;protein：0.3124±0.0472 VS 0.5320±0.0440,P0.01）.Additionally,mRNA and protein expressed by RASSF1A gene with methylated promotor reduced significantly than those expressed by RASSF1A gene with unmethylated promotor in cancer tissue（mRNA：0.0686±0.0174 VS 0.5511±0.0486,P0.0001;protein：0.1219±0.0326 VS 0.5614±0.0380,P0.0001）.Conclusion：Promotor methylation of RASSF1A gene in tissue of human colon carcinoma rises,which was correlated with the reduced RASSF1A gene transcription and expression and may lead to the functional deficit of RASSF1A gene in human colon carcinoma.