EGCG对肺癌细胞WIF-1基因启动子区的去甲基化作用  

Promoter demethylation of WIF-1 by epigallocatechin-3-gallate in lung cancer cells

作　　者：高志[1] 尤亮[1] 龚民[1] 崔永[1] 常栋[1] 马锴[1] 郑鑫[1] 王飞[1] 

机构地区：[1]首都医科大学附属北京友谊医院胸外科,北京100050

出　　处：《国际外科学杂志》2014年第3期188-192,共5页International Journal of Surgery

基　　金：北京自然科学基金（No.7102041）

摘　　要：目的 Wnt信号通路在许多恶性肿瘤包括肺癌的发生发展过程中发挥作用,WIF-1可以抑制Wnt通路的活性,但启动子区高甲基化导致WIF-1基因沉默,使Wnt信号通路异常激活并引起肿瘤.EGCG是一种去甲基化因子,本实验拟验证其去甲基化和对WIF-1基因的再激活作用.方法 利用甲基化特异性PCR,DNA测序及RT-PCR分析技术来测定WIF-1基因启动子区的去甲基化及WIF-1的表达,利用Westernblotting分析技术测定细胞质β-catenin蛋白的表达.结果 EGCG的浓度在0～50 μM时,在72 h培养期内对H460、A549及HCT116细胞系无细胞毒作用.在H460及A549细胞系内发现启动区域的甲基化.EGCG处理H460后WIF-1启动区域甲基化水平由77.6％降至27.6％ （P ＜0.05）,而处理A549后则由76.5％降至28.6％ （P ＜0.05）.使用EGCG后显示肺癌细胞系WIF-1启动子区去甲基化及WIF-1表达恢复.结论 EGCG在逆转WIF-1基因启动子甲基化并再激活WIF-1基因中有潜在的治疗作用.Objective Aberrant promoter methylation of Wnt inhibitory factor-1 （WIF-1 ） is a fundamental mechanism of epigenetic silencing in human cancers. Epigallocatechin-3-gallate （EGCG） has been reported to di- rectly reactivate several methylation-silenced genes. The promoter demethy^ation and reactivation of WIF-1 has not previously been reported. Methods Methylation-specific PCR, Sequencing analysis and RT-PCR analysis were performed to evaluate promoter demethylation of WIF- 1 and WIF- 1 expression, Western blot analysis and Transient transfection and luciferase reporter assay were performed to evaluate expression of cytosolic β-catenin protein and TCF reporter activity. Results we demonstrate promoter demethylation of WIF- 1 and restoration of W1F- 1 expres- sion after EGCG treatment in H460 and A549 cell lines. EGCG also down-regulated cytosolic β-catenin protein and decreased TCF reporter activity. Conclusion These data suggest the potential therapeutic use of EGCG for the re- versal of WIF- 1 promoter methylation.

关 键 词：EGCG WIF-1 去甲基化 肺肿瘤 基因 治疗结果 

分 类 号：R734.2[医药卫生—肿瘤]