肼苯哒嗪对人乳腺癌细胞株MDA-MB-231雌激素受体α去甲基化的影响

The Effect of Demethylation by Hydralazine on Estrogen Receptor α of Negative Human Breast Cancer Cell Line

目的探讨肼苯哒嗪对雌激素受体(ER)α阴性人乳腺癌细胞株MDA-MB-231 ERα基因启动子区CpG岛DNA甲基化的影响。方法体外细胞培养,按药物浓度分为4组:分别用0、5、10、20μmol/L的肼苯哒嗪作用ERα阴性人乳腺癌细胞株MDA-MB-231细胞96 h;按药物作用时间分为4组:分别于0、72、96、120 h 10μmol/L肼苯哒嗪作用ERα阴性人乳腺癌细胞株MDA-MB-231细胞,倒置光学显微镜下观察肼苯哒嗪对细胞生长的影响。各组提取MDA-MB-231细胞总RNA,用RT-PCR及图像分析检测肼苯哒嗪对MDA-MB-231细胞ERαmRNA表达的影响;提取MDA-MB-231细胞基因组DNA,纯化、修饰后用甲基化特异性聚合酶链反应(MSP)检测乳腺癌细胞株MDA-MB-231 ERα基因启动子区CpG岛DNA甲基化状态。结果与对照组比较,5、10、20μmol/L肼苯哒嗪组ER/β-actin光密度积分值比值均明显升高(P<0.05或P<0.01);72、96、120 h 10μmol/L肼苯哒嗪组ER/β-actin光密度积分值比值均明显升高(P<0.05或P<0.01)。5μmol/L肼苯哒嗪作用120 h后ERα基因呈部分去甲基化状态,而经10μmol/L肼苯哒嗪作用120 h后的ERα基因呈完全去甲基化状态。结论肼苯哒嗪可以逆转ERα阴性人乳腺癌细胞株MDA-MB-231 ERα基因启动子区CpG岛DNA甲基化状态,使沉默基因重新表达。

Objective To explore the demethylation effect of hydralazine on estrogen receptor （ER） α of MDA-MB-231,a negative human breast cancer cell line. Methods MDA-MB-231 cells were cultured in vitro and then were underwent grouping in two different ways. Grouping 1 ： cells were randomized into four groups, and exposed to various concentrations of Hydralazine （0,5,10, 20μmol/L respectively） for 96 hours ; Grouping 2 ： Again, MDA-MB-231 cells were randomized into four groups and exposed to 10 μmol/L Hydralazine for various lengths s of time（0,72,96, 120 h） respectively. Inversed optical microscope was used to observe the growth status of the cells. A total of RNA of each group was extracted and the expression of ERα was detected with reverse transcription polymerase chain reaction （RT-PCR） and image analysis. Genetic DNA of each group was extracted, purified and modified. DNA methylation status was determined by methylation specific polymerase chain reaction （MSP）. Results For 5, 10, and 20 μmol/L Hydralazine group,the ratio of ER to β-actin in respect of optical density was respectively significantly higher（P〈0.05 or P〈0.01） than that of 0 μmol/L group;Compared with 0 h group, the ratio of ER to β-actin on optical density in 72,96,120 h group was significantly higher（P〈0.05 or P〈0.01）. After 120-hour-long exposure to 5 μmol/L Hydralazine,promoter of ERa showed partial demethylation, in contrast with complete demethylation after exposure to 10 μmol/L Hydralazine for 120 hours. Conclusion Hydralazine could demethylate promoter of ERa and induced reexpression of the silenced gene on human breast cancer cell line MDA-MB-231.