Atrogin-1基因在贲门腺癌组织中的表达及其异常甲基化

Expression and aberrant methylation of atrogin-1 gene in gastric cardia adenocarcinoma tissuse

目的：探讨atrogin-1基因在贲门腺癌（gastric cardia adenocarcinoma，GCA）中的异常甲基化及表达，并分析其临床意义。方法： 选用河北医科大学第四医院2004—2008年间的贲门腺癌患者手术标本共139例，分别应用亚硫酸氢盐转换-甲基化特异性PCR（bisulfite conversion-methylation specific polymerase chain reaction，BS-MSP）、RT-PCR和免疫组织化学法检测贲门腺癌组织及相应癌旁（距癌灶边缘3～5 cm）组织中atrogin-1基因的甲基化、mRNA和蛋白表达情况，应用免疫组织化学法检测相应组织中Smad4蛋白的表达。结果： 贲门腺癌组织中atrogin-1基因启动子区的甲基化率[44.6%（62/139）] 显著高于癌旁组织[3.6%（5/139）]（χ^2=63.891，P=0.001），且atrogin-1基因的甲基化与TNM分期及肿瘤的组织学分化程度密切相关（χ^2=6.144, P〈0.05）。贲门腺癌组织中atrogin-1基因的mRNA和蛋白表达水平显著低于癌旁组织[（0.482 5±0.175 4） vs （0.896 9±0.290 1），t=10.62, P=0.01；34.5% vs 82.0%, χ^2=4.441，P=0.001]，且与其启动子区的甲基化状态之间有明显的相关性（r=-0.256，P=0.001）。贲门腺癌组织中Smad4蛋白表达的阳性率显著低于癌旁组织（46.0% vs 95.7%； χ^2=2.945，P=0.001），且与atrogin-1蛋白表达之间呈明显的正相关（r=0.604，P=0.001）。结论： Atrogin-1基因启动子区高甲基化导致的基因沉默可能是贲门癌组织中此基因表达降低的机制之一。

Objective ： To investigate the expression and aberrant methylation of the atrogin-1 gene in gastric cardia adenocarcinoma （GCA）. Methods： Tumor and normal tissue samples were collected from GCA patients （ n =139） undergoing surgical treatment in the Fourth Hospital of Hebei Medical University between 2004 and 2008. DNA methylation in the atrogin-1 promoter was analyzed by bisulfite conversion-methylation specific polymerase chain reaction （BS-MSP）, atrogin-1 mRNA abundance by RT-PCR, and density of immunoreactive signals for atrogin-1 and Smad4 by immunohistochemical staining. Results： The frequency of aberrant DNA methylation in the promoter region of atrogin-1 was significantly higher inGCA than in normal tissues （44.6% vs 3.6%, P 〈0.05）. The methylation status of atrogin-1 in tumor tissues was associated with TNM stage and the degree of histological differentiation of the tumor （ P 〈0.05）. The mRNA and protein levels of atrogin-1 were significantly decreased in tumor tissues as compared with normal tissues （0.482 5±0.175 4 vs 0.896 9±0.290 1 and 34.5% vs 82.0%, respectively, P 〈0.01）. there was a negative correlation between atrogin-1 methylation and atrogin-1 mRNA and protein levels （ r =-0.256， P 〈0.01）. Smad4 protein was detected in 46.0% of tumor tissues but in 95.7% of non-tumor tissue （ P 〈0.01） and the protein level of smad4 was positively associated with that of atrogin-1 （ r ＝0604， P 〈0.01）. Conclusion： Hypermethylation in the promoter region of the atrogin-1 gene and the resultant decrease in atrogin-1 protein synthesis may play an important role in the pathogenesis of gastric cardia adenocarcinoma.