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目的:分析MCF-7/Adr及MCF-7细胞mdr-1基因启动子甲基化和组蛋白乙酰化状态,初步探讨乳腺癌多药耐药的表观遗传机制。方法:用甲基化敏感PCR技术检测两个细胞系mdr-1基因启动子甲基化状态。实时定量PCR技术检测DNA甲基转移酶(DNA methyltransferases,DNMTs)mRNA及组蛋白去乙酰化酶(histone deacetylases,HDACs)mRNA的表达。光密度值法检测组蛋白H3和H4乙酰化水平。结果:MCF-7细胞mdr-1基因启动子呈现高甲基化,MCF-7/Adr细胞mdr-1基因启动子呈现低甲基化。与MCF-7细胞比较,MCF-7/Adr细胞DNMT1,DNMT3a及DNMT3bmRNA表达显著下降(P<0.05)。MCF-7/Adr细胞组蛋白H3和H4乙酰化水平较MCF-7细胞明显升高(P<0.01)。与MCF-7细胞比较,MCF-7/Adr细胞HDAC1,HDAC2,HDAC7及SIRT1mRNA的表达显著下降(P<0.01)。结论:mdr-1基因启动子低甲基化、组蛋白H3和H4高乙酰化、DNMTs mRNA及HDACs mRNA低表达可能是介导MCF-7/Adr细胞MDR形成的重要表观遗传学因素。
OBJECTIVE: To analyze the promoter methylation status and histone acetylation status of mdr-1 gene in MCF-7 / Adr and MCF-7 cells and to explore the epigenetic mechanism of multidrug resistance in breast cancer. Methods: The methylation status of mdr-1 gene promoter in two cell lines was detected by methylation-sensitive PCR. The expression of DNA methyltransferases (DNMTs) mRNA and histone deacetylases (HDACs) mRNA were detected by real-time PCR. Densitometry was used to detect histone H3 and H4 acetylation levels. Results: The promoter of mdr-1 gene in MCF-7 cells was hypermethylated and the promoter of mdr-1 gene in MCF-7 / Adr cells was hypomethylated. Compared with MCF-7 cells, the expression of DNMT1, DNMT3a and DNMT3b mRNA in MCF-7 / Adr cells were significantly decreased (P <0.05). The histone H3 and H4 acetylation levels in MCF-7 / Adr cells were significantly higher than those in MCF-7 cells (P <0.01). Compared with MCF-7 cells, the expression of HDAC1, HDAC2, HDAC7 and SIRT1 mRNA in MCF-7 / Adr cells was significantly decreased (P <0.01). CONCLUSION: The hypomethylation of mdr-1 promoter and the hyper-acetylation of histone H3 and H4, and the low expression of DNMTs mRNA and HDACs mRNA may be important epigenetic factors that mediate MDR formation in MCF-7 / Adr cells.