启动子区5'CpG岛去甲基化对人肠癌RKO细胞生物学表型的影响

目的:探讨DNA启动子区5'CpG岛甲基化状态与人肠癌RKO细胞生物学特征的关系.方法:应用特异性DNA甲基转移酶抑制剂-5-氮-2'-脱氧胞苷(5-Aza-2'-deoxycytidine,5-Aza-CdR)处理肠癌RKO细胞72小时,甲基化特异性PCR(methylation-specific PCR,MSP)及DNA测序法分析p16/CDKN2抑癌基因5'CpG岛甲基化状态;MTT、FCM、荧光染色及透射电镜检测启动子区去甲基化后对细胞生长、形态和细胞周期凋亡的影响.结果:肠癌RKO细胞p16/CDKN2基因5'CpG岛呈高甲基化状态;DNA甲基转移酶抑制剂(5-Aza-CdR)能较好地逆转启动子区胞嘧啶甲基化状态;CpG岛去甲基化后能明显地抑制肠癌细胞的生长,增加细胞群体倍增时间(P＜0.01),诱导肠癌细胞凋亡,并呈良好的量效依赖关系.结论:通过逆转CpG岛高甲基化能有效地抑制肠癌细胞增殖,为临床治疗大肠癌提供新的作用靶点.

Effects of Promoter Region 5'CpG Island Demethylation on the Biological Behavior of Human Colorectal Cancer RKO Cells in Vitro

OBJECTIVE To explore the relationship between the methylation status of the promoter 5'CpG island region and The biological behavior of human colorectal cancer RKO cells in vitro.METHODS RKO cells were treated with a selective DNA methyltransferase inhibitor-5-aza-2'-deoxycytidine (5-aza-CdR)for 72 h.Methylationspecific PCR(MSP),T-A cloning and DNA sequence analysis were used to determinate the 5'CpG island methylation status of the P16/CDKN2 tumor suppressor gene.Cell growth,morphological changes and apoptosis were analyzed by the MTT assay,flow cytometry.fluorescence staining and electron microscopy.RESULTS The 5'CpG island of the p16/CDKN2 tumor suppressor gene in RKO cells was a typically hypermethylated.The DNA methyltransferase inhibitor(5-Aza-CdR)effectively reversed the hypermethylation status of the promoter region.With demethylation,RKO cell growth was suppressed,the cells doubling times were prolonged(P＜0.01)and apoptosis was induced,which showed a relationship.CONCLUSION A selective DNA methyltransferase(DNMT)inhibitor can inhibit proliferation by demethylation in 5'CpG islands,and may be a potential new therapy targel for colorectal cancer.