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目的:应用甲基化特异性PCR(MSP)技术检测胃癌组织中Caveolin-1基因外显子2启动子区域5′端CpG岛甲基化状况,探讨Caveolin-1基因甲基化在胃癌发生发展中的作用.方法:收集30例胃癌组织和6例距肿瘤5cm以上的癌旁正常胃组织,运用标准蛋白酶K消化、酚-氯仿抽提法提取组织中基因组DNA,采用CpGenome DNA Modification Kit对DNA进行修饰后以甲基化特异性引物及非甲基化引物进行PCR,通过琼脂糖凝胶电泳及产物测序判定结果.结果:6例癌旁正常胃组织Caveolin-1基因外显子2启动子区域5′端CpG岛均为甲基化阴性.30例胃癌组织中有27例甲基化阳性,甲基化率为90%(27/30).其中16例胃癌组织(53.3%)仅有甲基化引物扩增出目的条带,表现为完全甲基化;11例胃癌组织(36.7%)甲基化与非甲基化引物均扩增出目的条带,表现为部分甲基化.统计结果显示胃癌组织中Caveolin-1基因外显子2启动子区域甲基化率显著高于癌旁正常胃组织.结论:胃癌组织中存在Caveolin-1基因外显子2启动子区域高甲基化,且可能参与胃癌的早期过程.
OBJECTIVE: To detect methylation status of CpG island at the 5’-end of Caveolin-1 gene promoter region in gastric cancer by methylation-specific PCR (MSP) technique and to explore the role of methylation of Caveolin-1 gene in the development of gastric cancer .Methods: Thirty gastric cancer tissues and 6 normal gastric tissues adjacent to tumor more than 5cm were collected. The genomic DNA was extracted by standard protease K digestion and phenol-chloroform extraction. The CpGenome DNA Modification Kit After modification, PCR was performed with methylation-specific primers and unmethylated primers, and the results were determined by agarose gel electrophoresis and sequencing.Results: The expression of Caveolin-1 gene exon 2 promoter in 6 adjacent normal gastric tissues Methylation was found in the 5 ’CpG islands of the region, methylation was found in 27 of 30 gastric carcinomas, and the methylation rate was 90% (27/30). Of the 16 gastric carcinomas (53.3%), only The methylation primers amplify the target bands and show complete methylation. The methylation and non-methylation primers of 11 cases of gastric carcinoma (36.7%) amplify the target bands and show partial methylation. The statistical results showed that the methylation rate of Caveolin-1 promoter region in gastric cancer tissue was significantly higher Which is higher than normal gastric tissue adjacent to cancer.Conclusion: Exon 2 promoter region of Caveolin-1 gene is highly methylated and may be involved in the early stage of gastric cancer.