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目的通过观察去甲基化药物5-Aza-Cd R对DU145前列腺癌细胞系RNF180基因的影响,探讨前列腺癌细胞系中肿瘤抑制基因RNF180失活的机制和原因。方法运用MTT法检测不同浓度(0、1、2、5、10、15、20μmo I/L)5-Aza-Cd R对前列腺癌细胞增殖能力的影响,同时筛选出1个最适药物浓度(5μmo I/L);分别采用Western blotting、实时PCR、甲基化特异性PCR(MSP)法检测最适药物浓度处理前后前列腺癌细胞中RNF180的表达情况。结果在一定范围内,5-Aza-Cd R对前列腺癌细胞DU145增殖能力的影响随着药物浓度的增加和药物处理时间的增长而加强(P<0.05);最适药物浓度处理后的前列腺癌细胞中RNF180蛋白及m RNA的表达量比处理前明显升高(P<0.05),而启动子区的甲基化程度明显降低。结论 5-Aza-Cd R能够逆转RNF180基因在DU145前列腺癌细胞中的高甲基化状态,解除RNF180基因表达沉默的状态。
Objective To investigate the effect of demethylation drug 5-Aza-Cd R on DU145 prostate cancer cell line RNF180 and investigate the mechanism and cause of inactivation of tumor suppressor gene RNF180 in prostate cancer cell lines. Methods The effects of 5-Aza-Cd R with different concentrations (0, 1, 2, 5, 10, 15 and 20μmo I / L) on the proliferation of prostate cancer cells were detected by MTT assay. 5μmo I / L). The expression of RNF180 in prostate cancer cells before and after the optimal drug concentration was detected by Western blotting, real-time PCR and methylation-specific PCR (MSP). Results In a certain range, the effect of 5-Aza-Cd R on the proliferation of prostate cancer cell DU145 was enhanced with the increase of drug concentration and drug treatment time (P <0.05). The optimal concentration of drug-treated prostate cancer The expression of RNF180 protein and m RNA in cells was significantly higher than that before treatment (P <0.05), while the methylation of promoter region was significantly decreased. Conclusions 5-Aza-Cd R can reverse RNF180 gene hypermethylation status in DU145 prostate cancer cells and release RNF180 gene silencing.