人肠癌RKO细胞周期依赖性激抑制因子基因启动子区甲基化状态的研究

目的 探讨人结肠癌RKO细胞周期依赖性激酶抑制因子(CKIs)家族启动子区CpG岛甲基化状态及其甲基化可逆性特征.方法 应用特异性DNA甲基转移酶(DNMTs)抑制剂5-Aza-2]-deoxycytidine(5-Aza-CdR)处理肠癌细胞,甲基特异性聚合酶链反应(Methylation-Specific PCR,MSP)、T-A克隆及DNA测序法分析RKO细胞CKIs家族抑癌基因p15ink4b、p16ink4a/CDKN2、p21/cip、p27/kip启动子CpG岛甲基化状态.结果 未经5-Aza-CdR作用的肠癌RKO细胞,其p15ink4b、p16ink4a/CDKN2基因组DNA胞嘧啶(C)保持不变,这提示在其单链DNA中胞嘧啶呈甲基化状态;而经5-Aza-CdR作用的肠癌RKO细胞,其p15ink4b、p16ink4a/CDKN2、p21/cip和p27/kip基因组DNA胞嘧啶均已变为胸腺嘧啶,表明在DNA单链中胞嘧啶已呈去甲基化状态.结论 肠癌RKO细胞周期INK4家族(p15ink4b和p16ink4a/CDKN2)基因的启动子区处于异常的高甲基化状态,而KIP/CIP家族(p21/cip和p27/kip)基因未处于高甲基化状态;5-Aza-CdR能较好地逆转肿瘤细胞INK4家族基因DNA高甲基化状态.

Methylation analysis of cyclin-dependent kinase inhibitor genes in human colorectal cancer cell

Objective To detect the methylation status of cyclin-dependent kinase inhibitor genes (CKIs)promoter region and their demethylation specifity in human colorectal cancer RKO cells.Methods RKO cells were treated with selective DNA methyltransferase enzyme(DNMTs)inhibitor-5.Aza-2'-deoxycytidine(5-Aza-CdR).Methylation-specific PCR(MSP),T-A clone and DNA sequence analysis were used to detect 5'CpG island methylation status of INK4(p15ink4b and p16ink4a/CDKN2)and CIP/KIP(p21/cip and p27/kip)promoter region respectively.Results All cytosines(C)of INK4(p15ink4b and p16ink4a/CDKN2)in the cells not treated witll 5-Aza-CdR RKO were remained C,which revealed all the cytosines in promoter region were methylated:However,all cytosines(C)of INK4(p15ink4b,p16ink4a/CDKN2),CIP/KIP(p21/cip,p27/kip)in the cells treated with 5-Aza-CdR were converted to T,which revealed all the cytosines(C)in promoter region were unmethylated.Conclusion INK4(p15ink4b and p16ink4a/CDKN2)promoter regions are in DNA abrreant hypermethylation in RK0 cells and the CIP/KIP (p21/cip and p27/kip)promoter regions arc not.The specific DNA 5-Aza-CdR may effectively cause demethylation of INK4(p15ink4b and p16ink4a/CDKN2).