地西他滨逆转人乳腺癌细胞中miR-5047基因甲基化状态的研究

Effect of methylation status of miR-5047 gene in breast cancer cells by Decitabine

目的探讨地西他滨对人乳腺癌细胞中miR-5047表达及其启动子甲基化状态的影响。方法用甲基化特异性PCR(MSP)法和qRT-PCR法分别检测人乳腺正常MCF-10A细胞和人乳腺癌MDA-MB-231、MCF-7细胞中miR-5047的表达水平及miR-5047启动子区的甲基化状态。用5μmol/L和10μmol/L浓度的地西他滨分别处理MDA-MB-231细胞,MSP法检测miR-5047启动子区的甲基化状态,qRT-PCR法检测miR-5047的表达。结果与人乳腺正常上皮细胞MCF-10A相比,乳腺癌MDA-MB-231和MCF-7细胞中miR-5047呈低表达,而其启动子区均呈高甲基化状态;地西他滨处理后,MDA-MB-231细胞中miR-5047启动子区的甲基化水平明显降低,miR-5047表达水平则显著升高。结论地西他滨可通过降低乳腺癌细胞中miR-5047基因启动子区甲基化水平从而促进miR-5047的表达。

Objective To investigate the effect of decitabine on the expression level of miR-5047 and promoter methylation status in human breast cancer cells.Methods Methylation specific PCR(MSP)and qRT-PCR were used to measure the methylation level of miR-5047 promoter and the expression of miR-5047 in human normal breast epithelial cells MCF-10A and human breast cancer MDA-MB-231 and MCF-7 cells.Then,MDA-MB-231 cells were treated with 5μmol/L and 10μmol/L of decitabine respectively,the methylation level of miR-5047 promoter was detected by MSP assay and the expression of miR-5047 was detected by qRT-PCR.Results Compared with MCF-10A cells,the expression level of miR-5047 in MDA-MB-231 and MCF-7 cells was lower,while the promoter region of miR-5047 in MDA-MB-231 and MCF-7 cells was hypermethylated.After decitabine treatment,the methylation level of miR-5047 promoter was markedly decreased and the expression level of miR-5047 was significantly increased in MDA-MB-231 cells.Conclusion Decitabine can promote the expression of miR-5047 by reducing the methylation level of miR-5047 promoter in human breast cancer MDA-MB-231 cells.