DR4去甲基化对髓性白血病K562细胞增殖凋亡作用的研究

Effect of DR4 Demethylation to the Proliferation and Apoptosis of Myeloid Leukemia K562 Cells

目的:研究肿瘤坏死因子死亡受体(DR)4去甲基化对髓性白血病K562细胞增殖、凋亡的影响。方法:取对数期K562细胞,采用地西他滨(DCA)0、0.8、1.6、3.2μmol/L干预,分别设为对照、DCA低剂量、DCA中剂量和DCA高剂量4组。取对照组细胞,采用肿瘤坏死因子相关凋亡诱导配体(TRAIL)0.5μg/ml干预24 h,设置为TRAIL组;取DCA高剂量组细胞,采用TRAIL 0.5μg/ml干预24 h,设置为DCA高剂量+TRAIL组。甲基化特异性聚合酶链反应(MS-PCR)检测对照组和DCA低、中、高剂量组DR4基因启动子甲基化状态;实时荧光定量聚合酶链反应(qRT-PCR)、Western blot检测对照组和DCA低、中、高剂量组DR4 mRNA及蛋白相对表达量;二甲基噻唑(MTT)法检测对照组、DCA高剂量组、TRAIL组、DCA高剂量+TRAIL组细胞增殖抑制率;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。结果:对照组细胞呈甲基化阳性状态,DCA低、中、高剂量组甲基化条带亮度逐渐降低至消失,DCA高剂量组呈甲基化阴性状态;对照组和DCA低、中、高剂量组DR4 mRNA及蛋白相对表达量依次升高(r=0.624、0.704);对照、DCA高剂量、TRAIL、DCA高剂量+TRAIL组24、48和72 h增殖抑制率、凋亡率依次升高(r=0.653、0.754、0.709,0.725)。结论:DCA可逆转ML K562细胞中DR4基因启动子甲基化水平,上调DR4表达,可能会增强TRAIL对K562细胞的增殖抑制及凋亡促进作用。

Objective:To investigate the effect of tumor necrosis factor death receptor(DR)4 demethylation to the proliferation and apoptosis of myeloid leukemia K562 cells.Methods:The logarithmic phase of K562 cells were treated by desitabine(DCA)at 0,0.8,1.6 and 3.2μmol/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)0.5μg/ml for 24 h,and the cells were divided into TRAIL group.The cells in DCA high dose group were treated by TRAIL 0.5μg/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.Realtime 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.Dimethylthiazole(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.Results: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.Conclusion: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.