上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

U937细胞系RFC1基因甲基化状态对细胞周期和凋亡的影响

张 娜，蒋 慧，李 红，邵静波，刘 青，杨静薇   

  1. 上海市儿童医院 上海交通大学附属儿童医院血液肿瘤科， 上海 200040
  • 出版日期:2015-01-28 发布日期:2015-01-29
  • 作者简介:张 娜(1980—)， 女， 主治医师， 硕士; 电子信箱: abczhangna@hotmail.com。
  • 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

ZHANG Na, JIANG Hui, LI Hong, SHAO Jing-bo, LIU Qing, YANG Jing-wei   

  1. Department of Hematology and Oncology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China
  • Online:2015-01-28 Published:2015-01-29
  • Supported by:

    Youth Research Program of Shanghai Municipal Health Bureau, 20114y068

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摘要：

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。

关键词: U937, DNA 甲基化, 还原叶酸载体基因, 5-杂氮胞苷, 甲氨蝶呤

Abstract:

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