U937细胞系RFC1基因甲基化状态对细胞周期和凋亡的影响
网络出版日期: 2015-01-29
基金资助
上海市卫生局青年科研项目(20114y068)
Effects of methylation of RFC1 gene of U937 cell line on cell cycle and apoptosis
Online published: 2015-01-29
Supported by
Youth Research Program of Shanghai Municipal Health Bureau, 20114y068
目的 探讨急性单核细胞样细胞系U937的还原叶酸载体基因(RFC1)启动子甲基化表达及其转录状态,并观察甲基化水平的改变对甲氨蝶呤(MTX)诱导的细胞凋亡和周期的影响。方法 运用定量甲基化特异性PCR和实时荧光定量PCR检测U937细胞系经5-杂氮胞苷作用后的启动子甲基化状态及其RFC1基因转录表达水平。通过流式细胞术验证RFC1基因表达变化对MTX诱导的U937细胞凋亡和细胞周期的影响。结果 U937细胞RFC1启动子的非甲基化率为(8.09±0.87)%,处于高甲基化状态;经1、2、4 μmol/L的5-杂氮胞苷作用72 h后,非甲基化率分别为(9.44±2.01)%、(13.51±2.19)%、(15.76±1.65)%;2、4 μmol/L的5-杂氮胞苷作用后,RFC1的非甲基化率较对照组明显升高(P=0.005, P=0.001)。2、4 μmol/L的5-杂氮胞苷处理后,RFC1 mRNA表达量上升,差异有统计学意义(P=0.003, P=0.000)。2 μmol/L 5-杂氮胞苷可抑制细胞S期,同时G1期细胞增加;5 nmol/L 的MTX引起细胞凋亡,并特异性作用于S期;两者联用可显著增加细胞凋亡,增强S期的抑制作用。结论 U937细胞系中RFC1基因启动子存在异常高甲基化,5-杂氮胞苷可引起去甲基化作用,并引起RFC1表达增加,从而增加MTX引起的细胞凋亡,特异性抑制细胞周期S期。
张 娜 , 蒋 慧 , 李 红 , 等 . U937细胞系RFC1基因甲基化状态对细胞周期和凋亡的影响[J]. 上海交通大学学报(医学版), 2015 , 35(1) : 41 . DOI: 11.3969/j.issn.1674-8115.2015.01.008
Objective To investigate the methylation, expression, and transcription of the promoter of reduced folate carrier 1 (RFC1) gene of U937 cell line and to observe the effects of variations of the methylation level on the cell apoptosis and cycle induced by methotrexate (MTX). Methods The quantitative real-time methylation-specific PCR and real-time quantitative PCR were adopted to detect the methylation status and mRNA expression of RFC1 gene promoter of U937 cell line after being incubated by 5-azacytidine. The effects of variations of RFC1 gene expression on the apoptosis and cell cycle of U937 cells induced by MTX were verified by the flow cytometry. Results The un-methylation rate of RFC1 gene promoter of U937 cells was (8.09±0.87)% which was in high methylation status and increased to (9.44±2.01)%, (13.51±2.19)%, and (15.76±1.65)% after being demethylated by 5-azacytidine of 1, 2, and 4 μmol/L for 72 h, respectively. Compared with the control group, the un-methylation rate of RFC1 promoter significantly increased (P=0.005, P=0.001) and the mRNA expression of RFC1 promoter increased (P=0.003, P=0.000) after being treated by 5-azacytidine of 2 and 4 μmol/L. And 5-azacytidine of 2 μmol/L inhibited the S phase and increased G1 phase of cell cycle. MTX of 5 nmol/L induced the apoptosis and inhibited the S phase of cell cycle. The combination of MTX and 5-azacytidine significantly increased the apoptosis and inhibited S phase of cell cycle. Conclusion The methylation level of RFC1 gene promoter of U937 cell line is abnormally high. And 5-azacytidine can demethylate and increase the mRNA expression of RFC1 gene promoter, therefore enhance the apoptosis effect of MTX and specifically inhibit the S phase of cell cycle.
Key words: U937; DNA methylation; reduced folate carrier gene; 5-azacytidine; methotrexate
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