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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。
OBJECTIVE: To investigate the relationship between methylation status of 5’CpG island in DNA promoter region and biological characteristics of human colorectal cancer RKO cells. Methods: Colorectal cancer RKO cells were treated with 5-Aza-2’-deoxycytidine (5-Aza-CdR), a specific inhibitor of DNA methyltransferase for 72 hours, Methylation status of 5’CpG island of p16 / CDKN2 tumor suppressor gene was analyzed by methylation-specific PCR (MSP) and DNA sequencing. The methylation status of 5’CpG island of p16 / CDKN2 tumor suppressor gene was analyzed by MTT, FCM, fluorescent staining and transmission electron microscopy. Effects of Cell Growth, Morphology and Cell Cycle Apoptosis. Results: The 5’CpG island of p16 / CDKN2 gene in colorectal cancer was hypermethylated and the DNA methyltransferase inhibitor (5-Aza-CdR) could reverse the cytosine methylation in promoter region. CpG island Demethylation could significantly inhibit the growth of colorectal cancer cells, increase the doubling time of cell population (P <0.01) and induce the apoptosis of colorectal cancer cells with good dose-response relationship. Conclusion: Hypermethylation of CpG island can effectively inhibit the proliferation of colorectal cancer cells and provide a new target for clinical treatment of colorectal cancer.