GADD45A基因在人贲门腺癌组织中的异常甲基化和表达及其临床意义

Expression and aberrant methylation of growth arrest and DNA-damage-inducible 45 alpha gene in human gastric cardia adenocarcinoma tissues and its clinical significance

目的:探讨生长阻滞和DNA损伤诱导45A(growth arrest and DNA-damage-inducible 45 alpha,GADD45A)基因在贲门腺癌(gastric cardia adenocarcinoma,GCA)中的异常甲基化及表达,并探讨其临床意义。方法:选取河北医科大学第四医院2004-2007年期间GCA患者组织标本(138例)。分别应用亚硫酸氢盐测序法(bisulfite sequencing,BS-Seq)、亚硫酸氢盐转换-甲基化特异性聚合酶链式反应(bisulfite conversion-methylation specific polymerase chain reaction,BS-MSP)、RT-PCR和免疫组织化学法检测GADD45A基因在GCA组织及癌旁正常组织中的甲基化、GADD45A mRNA及蛋白表达的情况。结果:GADD45A远端启动子区的4个CpG位点在GCA组织中的甲基化率[44.93%(62/138)]显著高于癌旁正常组织[0.00%(0/138)](P<0.01),且在Ⅲ期和Ⅳ期GCA组织中的甲基化率显著高于Ⅰ期和Ⅱ期GCA组织(P<0.05),但GADD45A在GCA组织中的甲基化与患者的年龄、性别及病理分化程度无关(P>0.05)。GADD45A近端启动子(region 2)及第一外显子区(region 3)的CpG岛在GCA及癌旁组织中均未检测到甲基化。GCA组织中GADD45A mRNA和蛋白阳性表达率显著低于癌旁正常组织[(0.35±0.15)vs(0.78±0.26),42.75%vs 71.01%,均P<0.05],且与其远端启动子区4个CpG位点的甲基化状态之间有明显的相关性(r=-0.52,P<0.01)。结论:GADD45A基因远端启动子区的4个CpG位点的高甲基化导致的基因沉默可能与GCA中GADD45A基因表达降低有关。

Objective：To investigate the expression and aberrant methylation of growth arrest and DNA-damage-inducible 45 alpha（GADD45A） gene in gastric cardia adenocarcinoma（GCA） and to explore its clinical significance.Methods： Tissue samples in GCA patients（138 cases） were selected from Fourth Hospital of Hebei University during 2004 to 2007.Bisulfite sequencing（BS-Seq）,bisulfite conversion-methylation specific polymerase chain reaction（BS-MSP）,reverse transcription-polymerase chain reaction（RT-PCR） and immunohistochemistry methods were used respectively to detect the methylation status,mRNA and protein expression of GADD45A gene in GCA tissues and the para-carcinoma normal tissues.Results： The methylation frequency of four CpG sites in distal promoter of GADD45A in GCA tissues（44.93%,[62/138]） was significantly higher than that in the para-carcinoma normal tissues（0.00%,0/138）（P0.01）.The methylation frequency of 4 CpG sites in stage Ⅲ and Ⅳ GCA tissues was significantly higher than that in stage Ⅰ and Ⅱ GCA tissues（P0.05）.However,the methylation status GADD45A in GCA tissues was not correltaed with age,gender and pathological differentiation（P0.05）.For GADD45A region 2 and 3 located in proximal promoter and exon 1,no methylation was detected in GCA and the para-carcinoma normal tissues.The expression of GADD45A mRNA and positive expression rate of GADD45A protein in GCA tissues were significantly lower than those in the para-carcinoma normal tissues（[0.35±0.15] vs [0.78±0.26],42.75% vs 71.01%,P0.05） and was associated with methylation status of 4 CpG sites in distal promoter（r=-0.52,P0.01）.Conclusion： Hypermethylation of four CpG sites in distal promoter of GADD45A gene may be responsible for the decreased expression of GADD45A in GCA.