贲门腺癌中Smad4基因甲基化状态分析

目的：探讨贲门腺癌（gastric cardia adenocarcinoma,GCA）中Smad4（mothers against decapentaplegic homolog4）基因启动子区及第1外显子区的甲基化状态及其与TGF-β1蛋白表达之间的相关性。方法：取110例贲门腺癌组织及相应癌旁组织,分别采用甲基化特异性PCR（methylation specific PCR,MSP）方法检测Smad4基因的甲基化情况,RT-PCR方法检测Smad4 mRNA水平,免疫组织化学法检测Smad4和TGF-β1的蛋白表达情况。结果：贲门腺癌组织Smad4基因启动子甲基化率为5.5%（6/110）,Smad4基因第1外显子5′非翻译区的甲基化率为24.5%（27/110）,相应癌旁正常黏膜均未检测到此两个位点的甲基化,癌组Smad4基因甲基化率显著高于癌旁正常组织（P〈0.05）,且贲门腺癌中Smad4基因5′非翻译区发生甲基化的比率显并高于启动子区（P〈0.01）。贲门腺癌组织中Smad4 mRNA及蛋白表达显著低于癌旁正常组织（P〈0.05）,其甲基化与蛋白表达之间呈负相关。TGF-β1在贲门癌组织中的表达（65.5%）明显高于相应癌旁组织（15.5%）（P〈0.01）,且随肿瘤分期的增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）。Smad4蛋白和TGF-β1蛋白在贲门腺癌中的表达呈明显的负相关。结论：在贲门腺癌发生发展过程中Smad4基因第1外显子的5′非翻译区比启动子区更易发生高甲基化而导致基因沉默,Smad4甲基化及TGF-β1的过表达可能是贲门腺癌发生的机制之一。

CpG island methylation of Smad4 gene in gastric cardia adenocarcinoma

OBJECTIVE:We investigated the promoter and exonl methylation of mothers against decapentaplegic homolog 4(Smad4) and its correlation with protein expression of TGF-β1 in gastric cardia adenocarcinoma(GCA). METHODS:Methylation-specific PCR approach,RT-PCR and immunohistochemistry methods were used to examine the methylation status of the 5’ CpG island,mRNA and protein expression of Smad4.Immunohistochemistry method was used to detect the protein expression of TGF-β1 in tumors and corresponding normal tissues.RESULTS:For the promoter site,Smad4 was methylated in 6/110(5.5%) tumor specimens.For the 5’ UTR of exonl,Smad4 was methylated in 24.5%(27/110) tumor specimens.Both were significantly higher than that in corresponding normal tissues(P<0.05). Methylation frequency of Smad4 in the 5’ UTR of exonl was significantly higher than that in the promoter site(P<0.01). Smad4 mRNA and protein expression of tumor tissues were significantly lower than that in corresponding normal tissues and was inversely correlated with its methylation status.Seventy-two of 110(65.5%) tumor tisssues demonstrated positive immunostaining for TGF-β1 and the frequency of positive TGF-β1 protein expression was significantly higher than that in corresponding normal tissues(P<0.01).The protein expressions of TGF-β1 were significantly different among various stages and differentiations of GCA(P<0.05).The protein expression of Smad4 was inversely correlated with TGF-β1 in GCA.CONCLUSION:Hypermethylation of Smad4 in 5’ UTR of exon1 and the higher protein expression of TGF-β1 may play important roles in the development of gastric cardia adenocarcinoma.