STAT1信号通路在全反式维甲酸诱导人白血病细胞向粒细胞分化过程中的作用与机制

Role and molecular mechanism of STAT1 signaling pathway in the differentiation of leukemia cells into granulocytes induced by all-trans retinoic acid

目的探讨信号转导与转录激活因子1(STAT1)信号通路在全反式维甲酸(ATRA)诱导人白血病细胞向粒细胞分化过程中的作用与机制,为白血病的临床治疗提供新的靶点与途径。方法将人急性髓系白血病细胞HL-60随机分为两组,观察组以ATRA(2μmol/L)诱导HL-60细胞向粒细胞分化,对照组加入相同浓度DMSO;瑞氏-吉姆萨染色观察细胞形态变化,流式细胞术检测细胞表面CD11b表达,q-PCR法检测细胞STAT1、亮氨酸结构拉链转录因子ε(C/EBPε)mRNA,Western blotting法检测细胞STAT1蛋白及磷酸化(p-STAT1)水平,q-PCR法检测细胞miR-155-5p,Pearson相关性分析STAT1与miR-155-5p、C/EBPε表达的关系。结果与对照组比较,观察组诱导96 h后细胞体积变小,形状不规则,胞质增多,核浓缩,核仁变小或消失,分叶核显著增多。观察组ATRA诱导96 h时细胞表面CD11b阳性表达率为62.97%±1.90%,高于对照组的1.87%±0.08%(P<0.05)。与对照组比较,观察组ATRA诱导后48、72、96 h细胞中STAT1 mRNA、C/EBPεmRNA、STAT1蛋白、p-STAT1蛋白表达增加(P均<0.05),而miR-155-5p表达降低(P均<0.05)。Pearson相关性分析显示,细胞STAT1 mRNA与miR-155-5p相对表达量呈负相关(r=-0.90,P<0.05),与C/EBPεmRNA相对表达量呈正相关(r=0.96,P<0.05)。结论miR-155-5p反向调控STAT1信号通路,进而活化粒细胞分化转录因子C/EBPε,诱导HL-60细胞向粒细胞分化;STAT1活化可能是ATRA诱导HL-60细胞向粒细胞分化的关键机制之一,靶向调控STAT1信号通路可能成为诱导HL-60细胞向粒系分化的有效靶点。

Objective To detect the role and regulatory mechanism of signal transducer and activator of transcription 1（ STAT1） in promoting the differentiation of human leukemia cells into granulocytes induced by all-trans retinoic acid（ ATRA）,and to provide a novel target for clinical treatment. Methods Human acute myeloid leukemia cells HL-60 were randomly divided into the control group and observation group. In the observation group,HL-60 cells were induced to differentiate into granulocytics with ATRA（ 2 μmol/L）,while cells in the control group were given the same concentration of DMSO. Morphologic changes of cells were observed by Wright-Giemsa staining. Cell surface marker CD11 b was detected by flow cytometry. The mRNA expression levels of STAT1 and CCAAT/enhancer binding proteinsε（ C/EBPε） were detected by q-PCR,the protein and phosphorylation levels of STAT1（ p-STAT1） were detected by Western blotting,and the transcriptional level of miR-155-5 p was detected by q-PCR. Pearson correlation analysis was used to detect the correlation of STAT1 with C/EBPε and miR-155-5 p. Results Compared with the control group,the HL-60 cells in the observation group became smaller and irregular,cytoplasm increased,nuclear concentrated,nucleolus became smaller or disappeared,and the number of leaf nuclei increased significantly. The rate of CD11 b positive HL-60 cells in the observation group at 96 h after ATRA induction was 62. 97% ± 1. 90%,which was higher than that in the control group（ 1. 87% ± 0. 08%）（ P〈0. 05）. Compared with the control group,the mRNA expression levels of STAT1 and C/EBPε,and the protein expression levels of STAT1 and p-STAT1 in the observation group at 48,72,and 96 h after ATRA induction increased（ all P〈0. 05）,while the expression of miR-155-5 p decreased（ P〈0. 05）. Pearson correlation analysis showed that STAT1 mRNA was negatively correlated with miR-155-5 p（ r =-0. 90,P〈0. 05）,while was positively correlated with C/EBPε mRNA（ r = 0. 96,P〈0. 05）. Conclusions The miR-155-5 p reversely regulates STAT1 signaling pathway,thus activates the transcription of C/EBPε,which leads to the differentiation of HL-60 cells into granulocytes. Activation of STAT1 signaling pathway may be one of the key mechanisms in the differentiation of HL-60 cells into granulocytes,and STAT1 may be taken as a novel target for clinical treatment.