散发性结直肠癌中hMLH1基因启动子区CpG岛MVPs图谱的绘制与分析

Mapping and analysis of the methylation variable positions profiles of hMLH1 promoter CpG islands in human sporadic colorectal carcinoma

目的 绘制散发性结直肠癌患者hMLH1基因启动子区CpG岛甲基化可变位点（methylation variable posi—tions，MVPs）图谱，分析MVPs图谱与hMLH1蛋白表达的关系，寻找CpG岛中影响蛋白表达的关键CpG位点。方法采用重亚硫酸盐测序方法检测患者hMLH1基因启动子区CpG岛甲基化状态；采用免疫组化方法检测相应蛋白表达水平。结果 CpG岛1（CGI—Ⅰ）甲基化阳性率为20％（6／30）；CpG岛2（CGI—Ⅱ）甲基化阳性率为13．33％（4／30）。hMLH1蛋白表达阳性率为50％（15／30）。经X^2检验，CGI—Ⅰ甲基化与hMLH1蛋白表达缺失存在显著性差异（P〈0．05）、CGI-Ⅱ甲基化与hMLH1蛋白表达缺失无差异（P〉0．05）。结论 CGI—Ⅰ甲基化可能导致hMLH1蛋白不表达，其中1～28CpG位点为导致hMLH1蛋白表达缺失的关键区域。

Objective To map the complete methylation status of the hMLH1 promoter in sporadic colorectal carcinoma and analyze the relationship between MVPs （methylation variable positions） of hMLH1 promoter and the expression of hMLH1. Methods Methylation status of hMLH1 promoter was measured by bisulfite sequencing, hMLH1 protein expression was detected by immunohistochemistry. Results Out of the 30 sporadic colorectal carcinoma specimens, the hypermethylation of CGIs （CpG islands） 1 was in 6 and that of CGIs 2 in 4. The hMLH1 protein was detected in 15 specimens. Chi square test showed the methylation of CGIs 1 was closely related to loss of hMLH1 protein expression （P 〈 0. 05 ） and the methylation of CGIs 2 was not related to loss of hMLH1 protein expression （P 〉 0. 05 ）. Conclusion In CGIs 1, CpG positions from 1 to 28 are the critical region that could influence the expression of hMLH1.