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目的探讨去甲基化药物5-氮杂-2’-脱氧脱苷(5-Aza-CdR)对子宫内膜癌中Ras相关区域家族1A(RASSF1A)基因甲基化的作用。方法取子宫内膜癌细胞株HEC-1-B细胞随机分组,用不同浓度(1.0×10-6,3.0×10-6,10.0×10-6 mol/L)的去甲基化药物5-Aza-CdR处理后,采用甲基化特异性PCR、实时定量PCR、Western blot、TUNEL等技术,分析空白对照组及实验各组子宫内膜癌细胞株HEC-1-B中RASSF1A基因启动子区域甲基化情况、mRNA表达水平、蛋白表达和细胞凋亡的情况。结果人子宫内膜癌细胞株HEC-1-B细胞中RASSF1A基因启动子区存在高甲基化,mRNA、蛋白低表达,以及细胞凋亡失控。不同浓度的5-Aza-CdR可逆转RASSF1A基因高甲基化状态,恢复mRNA、蛋白表达量,并可抑制HEC-1-B细胞的生长及诱导凋亡。结论子宫内膜癌异常甲基化RASSF1A基因作为治疗靶点,去甲基化药物5-Aza-CdR是基因治疗的有效途径。
Objective To investigate the role of demethylation drug 5-aza-2’-deoxy-glycoside (5-Aza-CdR) in methylation of Ras-related region family 1A (RASSF1A) gene in endometrial carcinoma. Methods The endometrial carcinoma cell line HEC-1-B cells were randomly divided into 5 groups. The cells were treated with different concentrations (1.0 × 10-6, 3.0 × 10-6 and 10.0 × 10-6 mol / L) of demethylation drug 5- After Aza-CdR treatment, methylation-specific PCR, real-time PCR, Western blot, TUNEL and other techniques were used to analyze the promoter region of RASSF1A gene in the blank control group and the experimental endometrial cancer cell line HEC-1-B Methylation, mRNA expression, protein expression and apoptosis. Results There was hypermethylation, low mRNA and protein expression of RASSF1A promoter in human endometrial carcinoma cell line HEC-1-B and uncontrolled apoptosis. Different concentrations of 5-Aza-CdR can reverse the hypermethylation status of RASSF1A gene and restore mRNA and protein expression, as well as inhibit HEC-1-B cell growth and induce apoptosis. Conclusion The abnormal methylation of endometrial cancer RASSF1A gene as a therapeutic target, demethylation drug 5-Aza-CdR is an effective way of gene therapy.