多基因启动子甲基化参与的乳腺癌MCF-7对他莫昔芬敏感性探究  

The sensitivity of breast cancer MCF-7 on tamoxifen and multiple gene promoter methylation status

作　　者：郑璐[1] 汤铜[1] 钱波[1] 李佳[1] 

机构地区：[1]安徽医科大学第二附属医院普通外科,合肥230601

出　　处：《安徽医科大学学报》2014年第12期1717-1721,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:1308085QH152)

摘　　要：目的探讨多基因启动子甲基化参与的乳腺癌MCF-7对他莫昔芬敏感性的关系。方法用MTT法对比乳腺癌MCF-7细胞株与耐药株生长增殖差异;流式细胞检测亲本与耐药株细胞凋亡之间的差异;RT-PCR及Real-Time PCR对比两细胞株相关基因mRNA表达差异;甲基化特异性PCR(MSP)检测4个候选基因(DAPK、MGMT、P53、RASSF1A)在亲本及耐药株中的甲基化状态。结果 Real-Time PCR可见MCF-7/TAM细胞中抑癌基因DAPK、RASSF1A水平低于亲本株(P<0.05);而MGMT、P53、DNMT1、DNM3B的mRNA表达水平却较亲本细胞株升高(P<0.05);基因DNMT3A的mRNA表达水平较低,在亲本及耐药株中均未检出。MSP显示,在MCF-7细胞中,DAPK、P53呈非甲基化状态,而对他莫昔芬细胞株这2个基因为甲基化状态。结合Real-Time PCR检测结果,基因甲基化状态与甲基转移酶(DNMT1、DNMT3B)表达呈正相关性;除P53基因外,基因mRNA表达水平与甲基化状态呈负相关性。结论相关基因表达差异及启动子的甲基化状态可能与乳腺癌细胞株对他莫昔芬敏感性相关,为下一步进行临床评估和机制探讨提供了依据。Objective To explore multiple gene promoter methylation status in breast cancer MCF-7 on the relationship between the tamoxifen sensitiity.Methods MTT method contrasted the proliferation differences between MCF-7 breast cancer cell lines and resistant strains.Flow cytometry（FCM） was used to detect the differences between parents and drug-resistant strains of apoptosis.RT-PCR and Real-Time PCR were used to compare two cell lines related genes mRNA expression differences.Methylation specific PCR （MSP） was used to detect the methylation status of 4 candidate genes （DAPK,MGMT,P53,RASSF1 A） in parents and resistant strains.Results The Real-Time PCR showed that the level of DAPK,RASSF1A were lower than the parent strain（P ＜ 0.05） ;while the expression level of MGMT,P53,DNMT1,DNM3B mRNA had increased compared to the parental cell line （P ＜ 0.05）.DNMT3A lower mRNA expression levels of genes in parental and resistant strains were not detected.MSP analysis showed that,DAPK,P53 were unmethylation status in MCF-7 cell,while these two genes were methylaed in resistant strains.Combined with the Real-Time PCR results,gene methylation status and methylatransferase （DNMT1,DNMT3B） expressed a positive correlation.In addition to the P53 gene,gene mRNA expression level was negatively correlated with methylation status.Conclusion The relative differences in gene expression and promoter methylation status may be associated with breast cancer cell lines of tamoxifen sensitiity,which will provide the basis for clinical evaluation and mechanism.

关 键 词：MCF-7 DNA甲基化 化疗耐受 抑癌基因 

分 类 号：R737.9[医药卫生—肿瘤]