DACT1基因不同区域异常甲基化对食管鳞癌患者预后的影响

Effect of aberrant methylation of DACT1 gene different regions on prognosis of the patients with esophageal squamous cell cancer

目的:检测食管鳞状细胞癌(esophageal squamous cell cancer,ESCC)组织标本及细胞株中β-环连蛋白抑制基因1(dishevelled-binding antagonist of beta-catenin 1,DACT1)Cp G岛旁区域及转录起始点(transcription start site,TSS)区域的甲基化状态,并进一步探讨DACT1基因两个不同区域甲基化状态对基因转录及预后的影响。方法:应用甲基化特异性PCR(methylation specific PCR,MSP)及RT-PCR的方法检测ESCC细胞株(TE1、TE13、T.Tn和Eca109)及河北省上消化道肿瘤高发区159例ESCC患者癌及相应癌旁组织中DACT1基因两个不同区域的甲基化状态及m RNA的表达情况。结果:DACT1基因在4株ESCC细胞系中均呈弱表达或阴性表达。应用甲基化抑制剂5-Aza-Dc处理该细胞株后,DACT1 m RNA表达明显增强;同时,MSP检测结果显示,此两区域的甲基化条带均明显减弱或消失;而应用组蛋白去乙酰化酶抑制剂TSA处理细胞株后,该基因在各细胞株中的表达无明显改变。DACT1 m RNA在ESCC组织中的表达较癌旁组织明显下调(P<0.01),且与该基因TSS区域异常甲基化状态有关(P<0.01);DACT1基因Cp G岛旁区域的甲基化频率在癌及相应癌旁组织中均较高(P>0.05),不具有肿瘤组织特异性,且对DACT1基因的转录抑制无明显影响(P>0.05);生存分析显示,DACT1基因TSS区域的甲基化状态与ESCC癌患者的生存期相关(P<0.01)。结论:ESCC中DACT1基因TSS区域的异常高甲基化状态是引起其表达下调的机制之一,并有望作为ESCC患者预后的甲基化标志物。

Objective： To investigate the methylation status of dishevelled-binding antagonist of beta-catenin 1 （DACT1） gene in CpG islands shore and transcription start site（TSS） regions in esophageal squamous cell cancer （ESCC） cell lines and ESCC samples, and to explore the possible effect on gene transcription and the prognosis of ESCC patients. Methods： MSP and RT-PCR methods were applied respectively to examine the methylation of DACT1 gene in two regions and its mRNAexpression in ESCC cell lines （TE1,TE13,T.Tn,Eca109） andESCC samples which from the high risk area of upper digestive tract cancer in Hebei Province. Results： The negative or weak expression of DACT1 mRNA was detected in four ESCC cell lines. After treated with 5-aza-2＇-deoxycytidine （5-aza-dC, a demethylation agent）, the expression level of DACT1 mRNA was obviously increased. Meanwhile, The result of MSP showed that the methylation bands were obviously weakened or disappeared. The level of DACT1 mRNA expression had no obviously change after treated with trichostatin A（TSA）. Decreased mRNA expression of DACT1 was observed in ESCC tumor tissues comparing with non-cancerous tissues（P〈0.01）, and associated with the methylation status of TSS region（P〈0.01）. The hypermethylation of DACT1 in CpG islands shore region was ob- served both in tumor and corresponding adjacent tissues but wasn’t related to the transcriptional inhibition of DACT1. The result of survival analysis showed that the methylation status of DACT1 in TSS region was associated with ESCC patients’prognosis（P〈0.01）. Conclusion： The highpermethylation of DACT1 in TSS region was one of the mechanisms causing genes silencing in ESCC and may serve as prognostic methylation biomarkers for ESCC patients.