化疗药物通过下调miR-148b对乳腺癌细胞ER-α甲基化影响

Chemotherapeutic drugs affect methylation of ER-α in breast cancer cells by down-regulating miR-148b

目的研究常用化疗药物对人乳腺癌细胞雌激素受体(estrogen receptor,ER)表达和甲基化的影响。方法采用剂量递增法,分别以紫杉醇(paclitaxel,PTX)和表柔比星(epirubicin,EPI)诱导>6个月,检测药物干预前后人乳腺癌细胞MCF-7(ER+,Luminal A)ER-α的表达和甲基化。用miRNA芯片筛选两种药物干预后MCF-7细胞中表达变化一致的miRNA,并以定量PCR(qPCR)验证。生物信息分析下调最显著的miRNA的靶点,将miRNA抑制剂(inhibitor)转染至MCF-7细胞,将miRNA模拟物(mimic)转染至MCF-7/PTX与MCF-7/EPI细胞,Western blot检测ER-α、DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)的表达情况,定量甲基化特异性PCR(qMSP)检测ER-α甲基化。结果建立对PTX耐药的MCF-7/PTX细胞株,对EPI耐药的MCF-7/EPI细胞株。药物干预后,均引起ER-α蛋白表达下降、甲基化水平升高,伴随DNMT1和组蛋白去乙酰化酶1(histone deacetylase 1,HDAC1)的表达上调,筛选出药物干预后MCF-7细胞中表达变化一致的miRNA,并经qPCR验证,其中下调最显著的是miR-148b。经生物信息学分析,通过荧光素酶报告基因实验(Luciferas)证实DNMT1是miR-148b的直接靶点。miR-148b inhibitor引起MCF-7细胞中ER-α表达降低、甲基化水平升高及DNMT1的表达增高;miR-148b mimic引起MCF-7/PTX与MCF-7/EPI细胞中ER-α表达升高、甲基化水平降低及DNMT1的表达降低。结论化疗药物(以PTX和EPI为代表)诱导MCF-7细胞中miRNA表达紊乱,下调的miR-148b对DNMT1表达的抑制作用减弱,进而促使ER-α发生高甲基化,表达下调。

Objective To investigate the effects of chemotherapeutic drugs on ER-α expression and methylation in breast cancer cells. Methods Human breast cancer cells MCF-7 (ER+, Luminal A) were induced by paclitaxel (PTX) and epirubicin (EPI) for more than 6 months, with an incremental dose, respectively. The expression and methylation status of ER-α in MCF-7 cells were detected before and after drug treatment. miRNAs with consistent expression changes in MCF-7 cells after two drugs’ treatment were screened by microarray, and verified by quantitative PCR (qPCR). Targets of the most significantly down-regulated miRNA were analyzed by bioinformatics. miRNA inhibitor was transfected into MCF-7 cells, miRNA mimic was transfected into MCF-7/PTX and MCF-7/EPI cells, then ER-α and DNA methyltransferase 1 (DNMT1) expression were detected by Western blot, and ER-α methylation was detected by quantitative methylation-specific PCR (qMSP). Results PTX resistant MCF-7/PTX cell line and EPI resistant MCF-7/EPI cell line were established. Both drug treatments caused a decrease in ER-α protein expression and an increase in methylation levels, with up-regulation of DNMT1 and histone deacetylase 1 (HDAC1) expression. miRNAs with consistent expression changes in MCF-7 cells after drug treatments were screened and verified by qPCR, the most significant down-regulation among which was miR-148b. Bioinformatics analysis, and further confirmed by luciferase reporter gene assay (Luciferas) that DNMT1 was a direct target of miR-148b. miR-148b inhibitor induced decreased expression of ER-α and increased methylation level in MCF-7 cells, accompanied by increased expression of DNMT1;whereas miR-148b mimic caused an increased expression of ER-α and decreased methylation level in MCF-7/PTX and MCF-7/EPI cells, with a decreased expression of DNMT1. Conclusion Chemotherapeutic drugs (represented by PTX and EPI) induce aberrant miRNA expression in breast cancer MCF-7 cells, and down-regulate miR-148b further to attenuate the inhibition of DNMT1 expression, which promote, hypermethylation and down-regulation of ER-α.