地西他滨对人急性髓系白血病细胞株HL-60的调控作用及其机制研究

Regulatory effect of decitabine on human acute myeloid leukemia cell line HL-60 and its mechanism

目的研究地西他滨（DAC）对人急性髓系白血病细胞株HL-60体外生长及自然杀伤（NK）细胞活化性受体配体（NKG2DL）表达的调节作用，并探讨JAK-STAT3-SOCS信号通路相关的分子机制。方法CCK-8法检测DAC对HL-60细胞增殖活性的影响，Annexin-V／PI双标法检测细胞凋亡，流式细胞术检测HL．60细胞表面NKG2DL分子MICA／B、ULBP的表达，羧基荧光素双乙酸盐（CFSE）法检测NK细胞的杀伤活性，蛋白印迹法分析细胞内JAK—STAT3通路中STAT3、STAT3上游激酶JAKl、JAK2及STAT3活性负调控因子细胞因子信号抑制物（SOCS）-1、SOCS-3的蛋白表达水平，甲基化敏感性高分辨率熔解曲线分析（MS-HRM）检测DAC处理后SOCS-1、SOCS．3基因甲基化程度。结果DAC可抑制HL-60细胞活性：0．2、0．5和1．0I．zmol／LDAC处理48h，HL-60细胞活性较对照组分别下降（25±11）％、（39±8）％和（50±7）％（P〈0．01）；48h时，细胞凋亡发生率分别为（24．77±7．50）％、（27．10±4．48）％和（30．53±3．93）％，均较对照组细胞的（3．11±0．50）％增加（P〈0．01）。DAC可诱导HL，60细胞表面MICA／B、ULBP-l及ULBP-3分子的表达增高，增强HL-60细胞对NK细胞的杀伤敏感性。DAC处理后HL-60细胞内STAT3、JAKl、JAK2及P-STAT3、p-JAKI、p-JAK2表达下降，SOCS-1和SOCS-3蛋白表达增高。DAC可抑制SOCS-3基因甲基化。结论DAC抑制人急性髓系白血病细胞株HL-60增殖，上调HL．60细胞对NKG2DL的表达，增强NK细胞对其的杀伤活性，其机制可能与细胞内JAK-STAT3-SOCS信号通路的活性调控有关。

Objective To investigate the effect of decitabine （DAC） on human acute myeloid leukemia （AML） cell line HL-60 and the regulating of natural killer （NK） cell activating receptor （NKG2D） ligands （NKG2DL）, and to detect the molecular mechanism of JAK-STAT3-SOCS signaling pathway. Methods The effect of DAC on the proliferation of HL-60 was detected by using CCK-8 assay. The cell apoptosis was analyzed by using Annexin-~/PI double standard method. The expressions of receptor NKG2DL including MICA/B and ULBPs in HL-60 cells were detected by using flow cytometry （FCM）. The killing activity of NK cells was analyzed by using earboxy fluorescein diacetate sueeinimidyl ester （CFSE）. The expressions of JAK/ STAT3 signaling pathway or molecules including STAT3, its upstream kinases JAK1, JAK2 and the negative regulator of STAT3, SOCS-1, SOCS-3 were examined by Western blot. Methylation level of the SOCS-1, SOCS-3 gene after the treatment of DAC was analyzed by using methylation-sensitive high resolution melting （MS-HRM）. Results There was an obvious inhibitory effect of DAC on HL-60 cells. The cell viability of HL-60 treated with 0.2, 0.5, and 1.0 ~mol/L DAC for 48 h was decreased by （25±11） %, （39＋8） % and （50±7） % （P 〈 0.01） respectively compared with those ceils without DAC treatment. The incidence of apoptosis was （24.77±7.50） %, （27.10±4.48） % and （30.53±3.93） % after DAC treatment for 48h respectively, which were higher than that of untreated cells [（3.11±0.50） %] （P 〈 0.01）. DAC induced a significant up-regulation of MICA/B, ULBP-1, ULBP-3 in HL-60 cells, and enhanced the sensitivity of HL-60 cells to NK cytotoxicity. Western blot results showed that a down-regulating expression of STAT3 and JAK1, JAK2 protein was detected, in addition to the phosphor-STAT3 and phosphor-JAKs in HL-60 cells after DAC treatment, but the expressions of SOCS-1 and SOCS-3 protein were increased. HRM results showed that DAC could inhibit the methylation of SOCS-3 gene. Conclusion DAC can inhibit the proliferation of HL-60 ceils, upregulate the expression of NKG2DL and enhance the cytotoxicity of NK targeted to HL-60 cells, which might be related to the activity regulation of intracellular JAK-STAT3-SOCS signaling pathway.