5-杂氮-2’-脱氧胞苷对人结肠癌细胞E-cadherin基因表达及甲基化的影响

Effect of 5-Aza-CdR on expression and methylation of E-cadherin gene in human colon carcinoma cells

背景与目的:结肠癌是最常见的恶性肿瘤之一,其发病机制仍未完全明了。目前认为DNA甲基化导致转录抑制是恶性肿瘤发生的重要机制之一。E-cadherin能抑制肿瘤的浸润和转移,被公认为是浸润、转移抑制基因。结肠癌常表现有E-cadherin基因失活。本研究通过检测人结肠癌细胞株HT-29中E-cadherin基因表达及甲基化状态,初步探讨结肠癌的发病机制。方法:光镜观察5-杂氮-2'-脱氧胞苷(5-Aza-CdR)干预前后细胞形态学变化;免疫细胞化学及RT-PCR检测不同浓度5-Aza-CdR干预对HT-29细胞中E-cadherin蛋白及mRNA表达的影响;甲基化特异性PCR(MSP)分析细胞中E-cadherin基因甲基化状态。结果:5-Aza-CdR干预能使其甲基化的E-cadherin基因重新表达;免疫细胞化学染色检测1μmol/L5-Aza-CdR干预24h后细胞E-cadherin阳性率由(21±7)%提高到5μmol/L5-Aza-CdR干预时的(39±13)%;E-cadherin基因在HT-29细胞中有甲基化修饰。结论:E-cadherin基因启动子区异常甲基化是结肠癌发生、发展的重要机制之一,5-Aza-CdR能使甲基化的E-cadherin去甲基化修饰并重新表达。

Background and Objective：Colon cancer is one of the most common malignant tumors,however,the pathogenesis of colon cancer is not fully clear.Transcriptional silencing induced by DNA methylation is believed to be an important mechanism of carcinogenesis.E-cadherin can suppress tumor cell invasion and metastasis,and is considered as an invasion and metastasis suppressor gene.Inactivation of E-cadherin gene often occurs in colon carcinoma.This study was to investigate the correlation between Ecadherin gene expression and the methylation status of E-cadherin 5＇ CpG islands in human colon carcinoma cell line HT-29,and to explore the mechanism of colonic carcinogenesis.Methods：Immunocytochemical dichostep method and RT-PCR were used to detect the expressions of E-cadherin protein and mRNA in HT-29 cells after treatment with different concentration of 5-Aza-CdR.Methylation specific polymerase chain reaction was used to analyze the methylation status at promoter of E-cadherin gene.Results：The expression of E-cadherin gene could be restored after treatment with 5-AzaCdR.Immunocytochemical staining showed the positive expression ratio of Ecadherin increased from （21±7）% （1 μmol /L） to （39±13）% （5 μmol /L） E-cadherin genes were methylated and not expressed in HT-29 cells.Conclusion：E-cadherin methylation may play an important role in the carcinogenesis of colon carcinoma cells and can re-express after the treatment with 5-Aza-CdR.