目的 观察去甲基化制剂5-氮-2'脱氧胞苷(5-aza-2'-deoxycytidine,5-Aza-dC)对人肺癌细胞系A549和LTEP-a2中CDH13基因启动子区甲基化状态及表达的影响,探讨肺癌细胞CDH13基因失活的机制及去甲基化制剂对CDH13基因表达的调控作用.方法 5-Aza-dC处理体外培养的肺癌细胞A549、LTEP-a2及正常肺细胞系HFL1后,用甲基化特异性 PCR( MSP) 法检测处理前后细胞中CDH13基因的甲基化状态,半定量逆转录-聚合酶链反应(RT-PCR)法检测用药前后细胞中CDH13 mRNA表达的变化.结果 正常肺细胞中未见CDH13启动子甲基化,肺癌细胞A549、LTEP-a2均发现CDH13启动子甲基化现象;应用 5-Aza-dC能使 CDH13基因启动子区 CpG 岛发生去甲基化.肺癌细胞A549、LTEP-a2均可见CDH13 mRNA表达,但与正常肺细胞比较表达较弱,经药物处理癌细胞后其CDH13 mRNA的表达较前明显增强.结论 启动子区异常甲基化是肺癌细胞CDH13基因失活的主要原因之一,去甲基化制剂5-Aza-dC能完全逆转CDH13基因甲基化状态,从而调控CDH13基因表达.%Objective To investigate the effects of 5-aza-2 '-deoxycytidine ( 5-Aza-Dc ) on methylation state and expression of CDH13 gene in human lung cancer cell lines A549 and LTEP-a2, and elucidate its underlying mechanisms. Methods The methylation specific PCR( MSP ) and RT-PCR was used to detect the changes of promoter methylation and Mrna expression of CDH13 gene in A549, LTEP-α2 and HFL1 cells before and after being incubated with DNA methyltransferase inhibitor 5-Aza-Dc, respectively. Results The promoter of CDH13 gene methylation was not detected in normal lung cell lines, but in both A549 and LTEP-a2 cells. Treatment with 5-Aza-Dc induced the demethylation of CpG islands in the promoter region. The Mrna expression of CDH13 gene was found in A549 and LTEP-a2 cells, but was weaker than that in normal cells. CDH13 Mrna expression was obviously enhanced in cancer cells treated with medicines. Conclusion Promoter abnormal methylation is a main cause of CDH13 gene inactivation in human lung cancer cell lines, which could be completely reversed by the demethylation agent 5-Aza-Dc.
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