贲门腺癌中TSP1启动子区高甲基化与TGF-β1表达的相关性分析

Correlation analysis of hypermethylation in the promoter region of TSP1 gene and the expression of TGF-β1 in gastric cardia adenocarcinoma

目的:探讨贲门腺癌中凝血酶敏感蛋白1(thrombospondin1,TSP1)基因的甲基化状态及其与转化生长因子β1(trans-forming growth factor-β1,TGF-β1)蛋白表达之间的相关性。方法:分别应用甲基化特异性PCR(methylation-specific PCR,MSP)、RT-PCR和免疫组织化学法检测贲门腺癌组织及相应癌旁组织的TSP1基因甲基化、mRNA和蛋白表达情况,应用免疫组织化学法检测对应组织中TGF-β1蛋白表达情况。结果:96例贲门腺癌组织中有34例发生了TSP1基因甲基化,甲基化发生率为35.4%,显著高于癌旁正常组织(P<0.001)。Ⅲ期和Ⅳ期贲门腺癌患者中TSP1基因发生甲基化的比率显著高于Ⅰ期和Ⅱ期患者(P<0.05)。贲门腺癌组织中TSP1 mRNA及蛋白表达显著低于癌旁正常组织(P<0.05),且与其甲基化状态之间有明显相关性。TGF-β1蛋白在贲门癌腺组织中的表达明显升高,96例贲门癌腺组织中有50例(52.1%)表达阳性,与相应癌旁正常组织相比差异有统计学意义(P<0.001);且随肿瘤分期的升高和肿瘤分化程度的降低,TGF-β1的阳性表达率明显升高(P<0.05)。TSP1基因在贲门腺癌中的高甲基化与TGF-β1蛋白表达之间有明显的相关性(P<0.05)。结论:TSP1基因启动子区高甲基化和TGF-β1蛋白过表达可能参与了贲门腺癌的发生、发展过程,TSP1基因启动子区发生甲基化导致的基因沉默可能是贲门腺癌发生的机制之一。

Objective：To investigate the methylation status of thrombospondin 1 （TSP1） gene and protein expression of transforming growth factor-β1 （TGF-β1） in gastric cardia adenocarcinoma （GCA）. Methods：Methylation-specific PCR （MSP） was used to detect the methylation status of TSP-1 gene,RT-PCR and Western blotting were used to determine the mRNA and protein expression of TSP1 in cardia adenocarcinoa tissues and pericancerous tissues,respectively. Immunohistochemistry method was used to detect the protein expression of TGF-β1 in GCA tissues and corresponding normal tissues. Results：TSP1 gene was methylated in 34/96 （35.4%） GCA specimens,which was significantly higher than that in corresponding normal tissues（P〈0.001）. The methylation frequencies of stage Ⅲ and Ⅳ GCA tissues were significantly higher than those in stageⅠandⅡ GCA tissues（P〈0.05）. TSP1 mRNA and protein expressions in GCA tissues were significantly lower than those in corresponding normal tissues （P〈0.05） and were correlated with the methylation status.TGF-β1 protein expression was significantly increased in GCA tissues,fifty out of ninty six （52.1%） gastric cardia adenocarcinoma tisssues demonstrated positive immunostaining of TGF-β1 and the frequency of positive protein expression of TGF-β1 was significantly higher than that in corresponding normal tissues（P〈0.001）. The protein expression of TGF-β1 was significantly increased with the increase in the TNM staging and decrease in the differentiation degree of GCA （P〈0.05）. Hypermethylation of TSP1 was correlated with the protein expression of TGF-β1 in GCA tissues（P〈0.05）.Conclusion：Promoter hypermethylation of TSP1 gene and overexpression of TGF-β1 protein are involved in the initiation and progression of gastric cardia adenocarcinoma. Gene silence induced by hypermethylation in the promoter region of TSP1 gene may be one of the mechanisms for oncogenesis of gastric cardia adenoma.