去甲基化修饰诱导人胆管癌细胞中RASSF1A基因表达

Re-expression of RASSF1A Gene by 5-Aza-CdR-induced Demethylation of the Promoter Region in Human Cholangiocarcinoma Cells

目的探讨DNA甲基转移酶抑制剂5-氮-2-脱氧胞苷(5-Aza-CdR)对人胆管癌细胞株QBC-939中RASSF1A基因启动子区域甲基化状态及其表达的影响。方法用5μmol/L的5-Aza-CdR对QBC-939细胞进行化学干预24 h,用甲基化特异性PCR(MS-PCR)检测RASSF1A基因启动子区域甲基化状态的改变,RT-PCR观察RASSF1A基因mRNA水平变化,Western blot检测RASSF1A蛋白的表达。结果经5-Aza-CdR化学干预后,RASSF1A基因启动子区域由甲基化状态转变为非甲基化状态;RT-PCR和Western blot结果显示在干预组细胞中分别出现RASSF1A基因的DNA条带(280 bp)和蛋白质条带(40 kD),而对照组中没有相应条带出现。结论DNA甲基转移酶抑制剂5-Aza-CdR能够诱导RASSF1A基因启动子区域去甲基化,并通过该机制诱导RASSF1A基因在人胆管癌细胞系QBC-939中的表达。

Objeetive To study the effect of DNA methyltransferase inhibitor 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on the methylation status in the promoter region of RASSF1A gene and its expression in human cholangiocarcinoma cell line QBC-939. Methods QBC-939 cells were treated with 5-Aza-CdR （5μmol/L） for 24 h. Methylation status in the promoter region of RASSF1A gene was detected by methylation-specific PCR （MS-PCR）, and the expression of RASSF1A mRNA and protein was observed by RT-PCR and Western blot respectively. Results After treatment with 5-Aza-CdR, methylaiton status in the promoter region of RASSF1A gene was reversed from methylation to un-methylation. A 280 bp DNA band that presented RASSF1A expression at transcriptional level and a 40 kD protein band that presented RASSF1A expression at protein level were detected by RT-PCR and Western blot respectively in the experimental group and there were no corresponding bands in the control group. Conclusion DNA methyltransferase inhibitor 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by methylation and induce the re-expression of RASSF1A in QBC-939.