胃癌形成过程中端粒酶反转录酶基因表达及启动子甲基化状态分析

Methylation status of the promoter and gene expression of telomerase reverse transcriptase during the carcinogenesis and progression of gastric carcinoma

目的探索端粒酶反转录酶(hTERT)基因表达情况及启动子区甲基化状态在胃癌形成过程中的变化情况。方法提取正常胃黏膜、胃癌前病变和胃癌3组组织样本的基因组DNA。用聚合酶链反应(PCR)特异性扩增其hTERT基因,用琼脂糖凝胶电泳检测3组组织样本中hTERT基因表达的阳性率;用亚硫酸氢盐修饰上述各组标本DNA,然后检测修饰后的hTERT序列:启动子区CpG位点,未发生甲基化的胞嘧啶(C)变为尿嘧啶(U);在启动子区不含CpG位点的位置,进行引物设计,然后利用PCR进行扩增。扩增产物序列与原序列比对,观察启动子区甲基化情况。结果 hTERT基因在胃癌组表达的阳性率最高,在癌前病变组表达的阳性率明显降低,而在正常胃黏膜组中不表达,3组组织样本hTERT表达阳性率比较差异均有统计学意义(P<0.05)。hTERT启动子区甲基化程度在胃癌组中最高,在胃癌前病变组中明显降低,而在正常胃黏膜中甲基化程度最低,3组组织样本hTERT启动子区甲基化程度比较差异均有统计学意义(P<0.05)。结论 hTERT启动子区甲基化程度在正常胃黏膜、胃癌前病变和胃癌中依次升高,故hTERT启动子区甲基化程度的变化可作为胃癌形成过程中肿瘤早期诊断的潜在分子生物学标志。

Objective To study the gene expression of human telomerase reverse transcriptase（hTERT） and the methylation status of its promoter during the carcinogenesis and progression of gastric carcinoma. Methods Genomic DNA was extracted from normal gastric mucosa samples, precancerous lesion samples and gastric cancer samples. The polymerase chain reaction（PCR） were carried to specific amplification the hTERT gene, and the agar gel electrophoresis was carried to identify the positive rate of the expression of hTERT gene in the three groups. Genomic DNA in each group was modified by sodium bisulrite, then the modified hTERT series were detected. The modification made anmethylated cytosine in the CpG sites in promoter region changed to uracil. The primers were designed in promoter region which did not include CpG site location and then it was amplified by PCR. The amplification product sequence was matched with the original sequence to observe the methylation status in promoter region. Results The positive expression rate of hTERT gene in the gastric carcinoma group was highest, and the rate in precancerosis group was decreased, and in normal gastric mucosa it did not express. The positive expression rate among three groups had significant difference（P 〈 0.05 ）. The methylation levels in promoter region of hTERT in the gastric carcinoma group were highest, and the levels in precancerosis group were decreased, and in normal gastric mucosa were lowest. The methylation levels among three groups had significant difference （ P 〈 0.05 ）. Conclusion The methylation levels in promoter region of hTERT in normal gastric mucosa, precancerous lesions and gastric carcinoma increased successively, so it may be a potential molecular biology maker in early diagnosis of gastric carcinoma.