摘要
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.
出处
《山东医药》
CAS
2018年第6期24-27,共4页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81373670
81573467)
山东省重点研发项目(2016GSF202016)
山东省自然科学基金资助项目(ZR2015HM014
ZR2017PH008)