Effects of suppressor of cytokine signaling 1 gene promoter demethylation on cell proliferation and apoptosis in myeloid leukemia cells
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摘要:目的 探讨髓系白血病(ML)细胞中细胞因子信号转导抑制因子1(SOCS-1)基因启动子去甲基化对细胞增殖、凋亡的影响。方法 收集78例ML患者的骨髓标本(实验组)以及人ML细胞系U937、HL-60、K562为研究对象，另外收集50例健康捐献者的骨髓标本作为对照组。甲基化特异性聚合酶链反应(MS-PCR)检测ML患者骨髓标本中SOCS-1基因启动子甲基化状态，实时荧光定量聚合酶链反应(qRT-PCR)检测ML患者骨髓标本以及人ML细胞系中SOCS-1 mRNA表达; Western blot检测ML患者骨髓标本以及人ML细胞系中SOCS-1蛋白表达。用0、0.8、1.6、3.2 μmol/L地西他滨(DCA)分别处理U937细胞，并分别命名为空白组、DCA-L组(DCA低剂量)、DCA-M组(DCA中剂量)、DCA-H组(DCA高剂量)。MS-PCR检测各组细胞中SOCS-1基因启动子甲基化状态; qRT-PCR检测各组细胞中SOCS-1 mRNA表达; Western blot检测各组细胞中SOCS-1蛋白表达; CCK-8法检测各组细胞增殖情况; 流式细胞术检测各组细胞凋亡情况。结果 SOCS-1在ML患者中呈高甲基化状态, SOCS-1 mRNA及SOCS-1蛋白在ML患者中的表达低于对照组，差异有统计学意义(P < 0.05); 人ML细胞系U937、HL-60、K562中SOCS-1 mRNA及SOCS-1蛋白表达低于人骨髓基质细胞HS-5, 差异有统计学意义(P < 0.05), 且U937细胞中SOCS-1 mRNA及SOCS-1蛋白表达水平最低，因此，以U937细胞为研究对象进行后续实验。与空白组比较, DCA-L组、DCA-M组、DCA-H组中SOCS-1甲基化水平、450 nm处光密度值(OD450 nm)(24、48、72 h)降低, SOCS-1 mRNA及SOCS-1蛋白表达水平、细胞凋亡率升高(P < 0.05), 且随着DCA浓度的升高，上述指标的相应趋势越明显。结论 SOCS-1去甲基化可抑制ML细胞增殖、促进细胞凋亡。
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关键词:
- 髓系白血病 /
- 细胞因子信号转导抑制因子1 /
- 甲基化 /
- 细胞增殖 /
- 细胞凋亡
Abstract:Objective To investigate the effects of demethylation of the promoter of suppressor of cytokine signaling 1 (SOCS-1) in myeloid leukemia (ML) cells on cell proliferation and apoptosis.Methods The bone marrow specimens of 78 ML patients (experimental group) and the human ML cell lines U937, HL-60 and K562 were collected as research objects, and bone marrow specimens from 50 healthy donors were collected as control group. Methylation-specific polymerase chain reaction (MS-PCR) was used to detect the methylation status of SOCS-1 gene promoter in bone marrow specimens of ML patients, quantitative real-time polymerase chain reaction (qRT-PCR)was used to detect the expression of SOCS-1 mRNA in bone marrow specimens of ML patients and human ML cell lines; Western blot was used to detect the expression of SOCS-1 protein in bone marrow specimens of ML patients and human ML cell lines. U937 cells were treated with 0, 0.8, 1.6 and 3.2 μmol/L Decitabine (DCA), and were selected as blank group, DCA-L group (DCA low-dose), DCA-M group (DCA medium-dose) and DCA-H group (DCA high-dose). MS-PCR was used to detect the SOCS-1 gene promoter methylation status in each group of cells; qRT-PCR was used to detect the SOCS-1 mRNA expression in cells of each group; western blot was used to detect the SOCS-1 protein expression in each group of cells; CCK-8 method was used to detect the cell proliferation in each group; flow cytometry was used to detect the cell apoptosis in each group.Results SOCS-1 was hypermethylated in ML patients, and the expressions of SOCS-1 mRNA and SOCS-1 protein in ML patients were significantly lower than that in the control group (P < 0.05); the expressions of SOCS-1 mRNA and SOCS-1 protein in human ML cell lines U937, HL-60, and K562 was significantly lower than that in human bone marrow stromal cells HS-5 (P < 0.05), and the expression levels of SOCS-1 mRNA and SOCS-1 protein in U937 cells were the lowest, therefore, the U937 cells were used as the research objects for follow-up experiments. Compared with the blank group, the SOCS-1 methylation levels and optical density 450 value (OD450 nm) at 24, 48 and 72 h in the DCA-L group, the DCA-M group and the DCA-H group were significantly reduced, and the SOCS-1 mRNA and SOCS-1 protein expression levels as well as the apoptotic rate were significantly increased (P < 0.05), and the corresponding trend of the above indicators became more obvious with the increase of DCA concentration.Conclusion SOCS-1 demethylation can inhibit the proliferation of ML cells and promote cell apoptosis. -
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表 1 SOCS-1基因启动子区域甲基化引物(SOCS-1 M)与非甲基化引物(SOCS-1 U)
引物 序列 产物大小/bp SOCS-1 M 正向: 5′-TTCGCGTGTATTTTTAGGTCGGTC-3′ 160 反向: 5′-CGACACAACTCCTACAACGACCG-3′ SOCS-1 U 正向: 5′-TTATGAGTATTTGTGTGTATTTTTAGGTTGGTT-3′ 175 反向: 5′-CACTAACAACACAACTCCTACAACAACCA-3′ 
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表 2 SOCS-1 mRNA及SOCS-1蛋白在ML患者和健康者中的表达水平(x±s)
组别 n SOCS-1 mRNA SOCS-1/β-actin 对照组 50 1.03±0.14 0.86±0.11 实验组 78 0.39±0.03* 0.27±0.02* 与对照组比较, *P < 0.05。
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表 3 SOCS-1 mRNA及SOCS-1蛋白在ML细胞系中的表达水平(n=6)(x±s)
细胞 SOCS-1 mRNA SOCS-1/β-actin HS-5细胞 1.04±0.11 0.87±0.12 U937细胞 0.38±0.04* 0.29±0.03* HL-60细胞 0.49±0.06* 0.36±0.07* K562细胞 0.56±0.07* 0.45±0.06* 与HS-5细胞比较, *P < 0.05。
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表 4 DCA对U937细胞中SOCS-1 mRNA及SOCS-1蛋白表达的影响(n=6)(x±s)
组别 SOCS-1 mRNA SOCS-1/β-actin 空白组 1.01±0.08 0.29±0.03 DCA-L组 1.31±0.13* 0.41±0.04* DCA-M组 1.61±0.18*# 0.58±0.06*# DCA-H组 1.95±0.24*#△ 0.89±0.09*#△ 与空白组比较, *P < 0.05; 与DCA-L组比较, #P < 0.05; 与DCA-M组比较, △P < 0.05。
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表 5 各组细胞OD450 nm值(24、48、72 h)比较(n=6)(x±s)
组别 OD450 nm值 24 h 48 h 72 h 空白组 0.63±0.05 1.18±0.18 1.55±0.15 DCA-L组 0.52±0.07* 0.85±0.14* 1.19±0.11* DCA-M组 0.41±0.04*# 0.63±0.12*# 0.84±0.12*# DCA-H组 0.27±0.06*#△ 0.39±0.03*#△ 0.47±0.06*#△ 与空白组比较, *P < 0.05; 与DCA-L组比较, #P < 0.05; 与DCA-M组比较, △P < 0.05。
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表 6 各组细胞凋亡率比较(n=6)(x±s)
组别 细胞凋亡率/% 空白组 1.56±0.23 DCA-L组 13.87±1.45* DCA-M组 25.72±3.24*# DCA-H组 39.56±4.61*#△ 与空白组比较, *P < 0.05; 与DCA-L组比较, #P < 0.05; 与DCA-M组比较, △P < 0.05。
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