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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 abnormal methylation and expression of growth arrest and DNA damage-inducible 45 alpha (GADD45A) gene in gastric cardia adenocarcinoma (GCA) significance. Methods: Tissue samples of GCA from the Fourth Hospital of Hebei Medical University from 2004 to 2007 were selected (138 cases). Bisulfite sequencing (BS-Seq), bisulfite conversion-methylation specific polymerase chain reaction (BS-MSP) and RT- PCR and immunohistochemistry were used to detect the methylation of GADD45A gene and the expression of GADD45A mRNA and protein in GCA tissues and adjacent normal tissues. Results: The methylation rates of four CpG sites in distal promoter region of GADD45A were significantly higher in GCA tissues (44.93% (62/138)] than in adjacent normal tissues [0.00% (0/138)] (P <0.01). The methylation rates of GCA tissues in stages Ⅲ and Ⅳ were significantly higher than those in stages Ⅰ and Ⅱ (P <0.05), but the methylation of GADD45A in GCA tissues was not related to the age, Sex and pathological differentiation was not related (P> 0.05). No methylation was detected in both GCA and paracancerous tissues by GADD45A proximal promoter region 2 and CpG island of the first exon region 3. The positive rate of GADD45A mRNA and protein expression in GCA tissues was significantly lower than that in the adjacent normal tissues [(0.35 ± 0.15) vs (0.78 ± 0.26), 42.75% vs 71.01%, both P <0.05] There was a significant correlation between the methylation status of CpG sites (r = -0.52, P <0.01). CONCLUSION: Gene silencing induced by hypermethylation at the four CpG sites in the distal promoter region of GADD45A gene may be related to the decrease of GADD45A gene expression in GCA.