【摘要】 目的探讨分泌型 Wnt 拮抗基因启动子 CpG 岛甲基化在结直肠肿瘤发生中的作用。方法 5-氮杂2′-脱氧胞苷(DAC)和曲古菌素 A(TSA)对结直肠癌细胞株 HCT116、SW480进行去甲基化处理。甲基特异性 PCR 和逆转录 PCR 分别检测结直肠肿瘤组织及细胞株中分泌型卷曲相关蛋白(sFRP)和 Wnt 抑制因子1(WIF-1)基因甲基化和 mRNA 表达。结果正常大肠黏膜不存在 sFRP和 WIF-1基因甲基化。sFRP1、2、4、5和 WIF-1在结直肠腺癌甲基化率分别为93.1%(67/72)、83.3%(60/72)、36.1%(26/72)、52.8%(38/72)、84.7%(61/72);腺瘤中分别为87.9%(29/33)、81.8%(27/33)、24.2%(8/33)、57.6%(19/33)、72.7%(24/33);瘤旁正常组织中分别为52.6%(20/38)、28.9%(11/38)、2.6%(1/38)、18.4%(7/38)、23.7%(9/38);与正常黏膜及瘤旁正常组织比较,差异均有统计学意义(均 P<0.05)。变性隐窝灶(ACF)中 sFRP1、2、4和5甲基化率分别为94.4(17/18)%、77.8%(14/18)、27.8%(5/18)和55.6%(10/18)。sFRP 和 WIF-1基因甲基化与结直肠腺癌、腺瘤的临床病理特征无关(P>0.05)。HCT116细胞株 sFRP1、2、4、5和 WIF-1基因启动子 CpG岛均存在高甲基化。SW480细胞株中仅 sFRP1、2和 WIF-1存在甲基化。甲基化的基因 mRNA 不表达或降低;联合使用 DAC 和 TSA 能有效恢复 sFRP 和 WIF-1基因表达。结论 Wnt 拮抗基因甲基化是结直肠肿瘤发生常见的早期事件,sFRP1、2、5和 WIF-1甲基化有可能成为早期发现结直肠肿瘤的生物学标志。更多还原

【Abstract】 Objective To investigate the functions of promoter hypermethylation of secreted Wnt- antagonist genes in colorectal tumorigenesis and progression.Methods Two colorectal cancer cell lines, HCT116 and SW480,were treated by 5-aza-2’-deoxycytidine(DAC)and trichostatin A(TSA)for demethylation.The promoter hypermethylation and expression of sFRP and WIF-1 genes in different stages of colorectal tumor and colorectal cancer cell lines were detected by methylation-specific PCR and reverse transcription PCR,respectively.Results None of the normal colorectal mucosa samples showed methylated bands of any sFRP and WIF-1genes.Hypermethylation of sFRP1,2,4,5 and WIF-1 was detected in 93.1%(67/72),83.3%(60/72),36.1%(26/72),52.8%(38/72)and 84.7,%(61/72)of adenocarcinomas,87.9%(29/33),81.8%(27/33),24.2%(8/33),57.6%(19/33)and 72.7,% (24/33)of adenomas,52.6%,28.9,%,2.6%,18.4%,23.7,% of the adjacent normal mucosa. Methylation was more frequently found in colorectal tumors than in normal mucosa and adjacent normal mucosa from patients with tumor(P<0.05).No significant association between Wnt-antagonist genes hypermethylation and clinicopathological characteristics was found(P>0.05).SFRP1,2,4,5 and WIF-1 genes were methylated in HCT116 cell line.SFRP1,2 and WIF-1 were methylated in SW480 cell line.The mRNA expression of sFRPs and WIF-1 genes was absent or significantly downregulated(P<0.01)when they were methylated in two colorectal cancer cell lines.SFRP3 was expressed in two colorectal carcinoma cell lines.DAC/TSA combination treatment re-expressed the silenced sFRPs and WIF-1 genes mRNA expressions effectively.A single application of TSA could not re-express sFRPs and WIF-1 genes mRNA expressions.The influence of demethylation treatment on sFRP3 expression was minimal.Conclusion Hypermethylation of Wnt-antagonist genes is a common early event in the evolution of colorectal tumor. Methylation of sFRP1,2,5 and WIF-1 genes might serve as biomarkers for the early detection of colorectal tumor.更多还原