Zhang Man, Cai Lin-Heng, Yang Hai-Ping, Yang Xue-Wen, Si Xiao-Hui
Department of Hematology, The First Affiliated Hospital of Henan University of Science and Technology, Luoyang 471000, Henan Province, China.
Department of Hematology, The First Affiliated Hospital of Henan University of Science and Technology, Luoyang 471000, Henan Province, China,E-mail：
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2021 Apr;29(2):422-427. doi: 10.19746/j.cnki.issn.1009-2137.2021.02.018.
To investigate the effect of tumor necrosis factor death receptor (DR) 4 demethylation to the proliferation and apoptosis of myeloid leukemia K562 cells.
The logarithmic phase of K562 cells were treated by desitabine (DCA) at 0, 0.8, 1.6 and 3.2 μmol/L, and the cells were divided into control group, DCA low dose group, DCA medium dose group and DCA high dose group respectively. The cells in control group were treated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 0.5 μg/ml for 24 h, and the cells were divided into TRAIL group. The cells in DCA high dose group were treated by TRAIL 0.5 μg/ml for 24 h, and were divided into DCA high dose + TRAIL group. Methylation-specific polymerase chain reaction (MS-PCR) was used to measure the methylation status of the DR4 gene promoter in the control group and DCA low, medium and high dose groups. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to determine the relative expression of DR4 mRNA and protein in the control group and DCA low, medium and high dose groups. Dime- thylthiazole (MTT) method was used to determine the inhibition rate of cell proliferation of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group. Flow cytometry was used to determine the apoptotic rate of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group.
The cells in the control group were methylation-positive, the brightness of the methylation bands of the cells in the DCA low, medium, and high dose groups was gradually decreased to disappear, and the DCA high dose group showed negative for methylation. The relative expression of DR4 mRNA and protein in the control group, DCA low, medium and high dose groups was increased sequentially (r=0.624, 0.704). The inhibition rate of cell proliferation of the cells in the control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group was increased sequentially (r=0.653, 0.754, 0.709, 0.725) at 24, 48 and 72 h.
DCA can reverse the methylation level of DR4 gene promoter in ML K562 cells and up-regulate the expression of DR4, which may enhance the proliferation inhibition and apoptosis promotion effects of TRAIL on K562 cells.
探讨肿瘤坏死因子死亡受体（DR）4去甲基化对髓系白血病K562细胞增殖和凋亡的影响。
将处于对数生长期的K562细胞分别用0、0.8、1.6和3.2 μmol/L的地西他滨（DCA）处理，细胞分别分为对照组、DCA低剂量组、DCA中剂量组和DCA高剂量组。对照组细胞用0.5 μg/ml肿瘤坏死因子相关凋亡诱导配体（TRAIL）处理24 h，分为TRAIL组。DCA高剂量组细胞用0.5 μg/ml TRAIL处理24 h，分为DCA高剂量+TRAIL组。采用甲基化特异性聚合酶链反应（MS-PCR）检测对照组及DCA低、中、高剂量组DR4基因启动子的甲基化状态。采用实时荧光定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法（Western blot）检测对照组及DCA低、中、高剂量组DR4 mRNA和蛋白的相对表达量。采用噻唑蓝（MTT）法检测对照组、DCA高剂量组、TRAIL组、DCA高剂量+TRAIL组细胞的增殖抑制率。采用流式细胞术检测对照组、DCA高剂量组、TRAIL组、DCA高剂量+TRAIL组细胞的凋亡率。
对照组细胞甲基化呈阳性，DCA低、中、高剂量组细胞甲基化条带亮度逐渐降低至消失，DCA高剂量组甲基化呈阴性。对照组、DCA低、中、高剂量组DR4 mRNA和蛋白的相对表达量依次升高（r=0.624，0.704）。对照组、DCA高剂量组、TRAIL组、DCA高剂量+TRAIL组细胞在24、48和72 h时的增殖抑制率依次升高（r=0.653，0.754，0.709，0.725）。
DCA可逆转ML K562细胞中DR4基因启动子的甲基化水平，上调DR4表达，可能增强TRAIL对K562细胞的增殖抑制和促凋亡作用。
