表观遗传学药物影响乳腺癌细胞系抑癌基因BRCA1及CHD5表达的研究

Effects of epigenetic drugs on BRCA1 and CHD5 expressions in breast cancer cells

目的明确表观遗传学药物对乳腺癌细胞系抑癌基因BRCA1及CHD5表达的作用,探讨此类药物在乳腺癌临床治疗中的合理应用。方法采用去甲基化制剂5-AZA和组蛋白去乙酰化酶抑制剂TSA分别作用于乳腺癌细胞系T47D、MCF-7及乳腺正常上皮细胞HBL-100,5-AZA终浓度分别为0、0.5、1.0、2.5、5.0和10.0μmol/L,TSA终浓度分别为0、0.25、0.5、0.75、1.0和1.5μmol/L。荧光定量RT-PCR检测各浓度5-AZA作用12、24、48及72 h,各浓度TSA作用6、12、24、48及72 h BRCA1和CHD5的表达水平,并行甲基化特异PCR(MSP)检测两基因的甲基化状态。结果 2.5μmol/L AZA和0.5μmol/L TSA作用可提高乳腺癌细胞系BRCA1及CHD5的表达,而10μmol/L AZA和1.0、1.5μmol/L TSA作用反致表达降低,且对乳腺正常上皮细胞呈毒性作用。MSP未能发现AZA和TSA对MCF-7细胞两抑癌基因及T47D细胞BRCA1甲基化状态的影响,而两种药物均可逆转T47D细胞CHD5的甲基化状态。结论表观遗传学药物对乳腺癌细胞抑癌基因表达的影响具有给药剂量及时间限制性,本研究可为此类药物在乳腺癌治疗中的科学应用提供实验依据。

Objective To investigate effects of epigenetic drugs on expressions of breast cancer susceptibility gene1（BRCA1） and chromodomain helicase DNA-binding protein（CHD5） in breast cancer cells,and explore the proper use of these drugs in breast cancer therapy.Methods MCF-7,T47D and HBL-100 cells were exposed to 5-azacytidine（5-AZA） of 0,0.5,1.0,2.5,5.0 and 10.0 μmol/L for 12,24,48 and 72 h,and trichostatin A（TSA） of 0,0.25,0.5,0.75,1 and 1.5 μmol/L for 6,12,24,48 and 72 h.After treatment,BRCA1 and CHD5 expressions were determined by quantitative RT-PCR,and methylation status of both genes was determined by methylation-specific PCR（MSP）.Results 2.5 μmol/L of 5-AZA and 0.5 μmol/L of TSA could improve expressions of BRCA1 and CHD5 in breast cancer cells,while drugs with too high doses,10 μmol/L of AZA and 1 and 1.5 μmol/L of TSA could decrease both gene expressions and were even toxic to normal cells.MSP showed no alterations in methylation status of BRCA1 in either cell line or CHD5 in MCF-7 after treatment,while both agents could reverse methylation status of CHD5 in T47D.Conclusion Epigenetic drugs have a dose-and time-dependent influence on tumor suppressor gene expression,and the present study provides fundamental experimental data for designed epigenetic therapy of breast cancer.