5-Aza-CdR对DU145前列腺癌细胞系RNF180基因去甲基化作用的初步研究

Preliminary Study on the Effect of 5-Aza-CdR on the Demethylation of RNF180 in Prostate Cancer Cell Line DU145

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 mechanism and cause of the inactivation of tumor suppressor gene RNF180 in prostate cancer Cell line by observing the effect of 5-Aza-CdR on the RNF180 gene in prostate cancer cell line DU145. Methods MTT method was adopted to study the effect of 5-Aza-CdR （0, 1, 2,5,10,15 and 20 μmol/L）on the proliferation of prostate cancer cells. Western blotting, real-time PCR, and methylation specific PCR （MSP） were separately used to detect the expression of RNF180 in prostate cancer cells before and after the treatment of the most suitable drug concentration （5 μmol/L）. Results In a certain range, the effect of 5-Aza-CdR on the proliferation of prostate cancer cell line DU145 was increased with the increase of drug concentxation and the time of drug treatment （P 〈 0.05）. After the treatment of the most suitble drug concentration, the protein and mRNA expression of RNF180 in prostate cancer cells was significantly increased （ P 〈 0.05 ）, but the methylafion of the promoter region was obviously decreased. Conclusion 5-Aza-CdR can reverse the methylafiou status of RNF180 gene in DU 145 prostate cancer cell line, and relieve the silencing status of RNF180 geue expression.