摘要
目的研究5-氮-2′-脱氧胞苷（5-Aza-CdR）对乳腺癌MCF-7细胞增殖及抑癌基因RASSF2A表达的影响。方法用不同浓度的5-Aza-CdR处理MCF-7细胞12-96h,四甲基偶氮唑盐比色（MTT）法和流式细胞仪分别检测药物处理前后细胞的增殖活性和凋亡率;实时荧光定量PCR（RT-qPCR）法检测各阶段RASSF2A mRNA的表达水平,甲基化特异性PCR（MSP）观察该基因甲基化的改变。结果用1、5、10μmol/L 5-Aza-CdR处理MCF-7细胞24、48、72和96h后,细胞生长均受到抑制,呈时间和剂量依赖性。流式细胞仪分析表明,3种药物浓度处理细胞72h后凋亡率增加,5和10μmol/L组凋亡率分别为（25.5±3.5）%和（58.2±3.2）%,与对照组（13.4±2.0）%相比差异有统计学意义（P〈0.05）。RT-PCR检测显示,不同浓度药物处理MCF-7细胞后,RASSF2A mRNA表达水平随着药物浓度的增加而逐步上调;进一步MSP检测发现这些癌细胞中RASSF2A甲基化状态得到了不同程度的逆转。结论 5-Aza-CdR可通过逆转RASSF2A基因异常甲基化导致该基因表达上调来抑制MCF-7细胞增殖,RASSF2A是一个乳腺癌相关抑癌基因。
Objective To investigate the effects of 5-Aza-2′-deoxycytidine（5-Aza-CdR）on the proliferation of MCF-7cells and the expression of RASSF2 A,a tumor suppressor gene.Methods The proliferation and apoptosis of MCF-7cells were examined by using MTT and flow cytometry（FCM）after they was treated with different concentrations of 5-Aza-CdR for 12-96 h.The expression of RASSF2 A mRNA was detected in MCF-7cells treated by 5-Aza-CdR for different time by real-time quantitative PCR（RT-qPCR）,and the methylation status of RASSF2 A was determined using methylation-specific polymerase chain reaction（MSP）.Results The proliferation of MCF-7cells was inhibited in a dose-and time-dependent manner after treatment with 1,5,and 10μmol/L 5-Aza-CdR for 24,48,72 and 96h,respectively.FCM showed the apoptosis rate was increased in MCF-7cells treated with 1,5,and 10μmol/L 5-Aza-CdR for 72 h.The apoptosis rate was（25.5±3.5）%in 5μmol/L 5-AzaCdR group and（58.2±3.2）% in 10μmol/L 5-Aza-CdR group,significantly higher than that in the control cells[（13.4±2.0）%,P〈0.05].RT-PCR revealed that the expression of RASSF2 A mRNA was increased with the concentration of 5-AzaCdR.Further MSP analysis demonstrated that the methylation status of RASSF2 A was reversed by 5-Aza-CdR to some degree.Conclusion 5-Aza-CdR may inhibit the proliferation of MCF-7cells by reversing the abnormal methylation status of RASSF2 Aand thus up-regulating its expression.RASSF2 Ais a tumor suppressor gene associated with breast cancer.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2015年第2期152-155,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.81272470)
江苏省六大人才高峰资助项目(No.2010-WS-075)
