miR-let-7c-3p靶向Egr-1调控白血病细胞定向单核/巨噬细胞分化的机制研究

Mechanism of miR-let-7c-3p targeting Egr-1to regulate monocyte/macrophage-directed differentiation of leukemia cells

目的探讨miR-let-7 c-3 p/早期生长反应因子-1(Egr-1)信号轴在白血病细胞定向单核/巨噬细胞分化中的作用与分子机制.方法人慢性髓系白血病K562细胞分别用100μg/L佛波酯(PMA,实验组)和0μg/L PMA(对照组)溶液向单核/巨噬细胞定向诱导分化48 h后,通过细胞形态学观察和流式细胞仪测定分化抗原,确定分化模型的成功建立;采用实时定量聚合酶链反应(qRT-PCR)检测白血病细胞分化前后Egr-1和miR-let-7c-3p的表达变化,蛋白质印迹法检测Egr-1蛋白表达变化;siRNA干扰实验检测Egr-1对细胞分化的影响,设PMA+si-Egr-1组和PMA+si-Ctrl组;双荧光素酶结合实验检测miR-let-7 c-3 p与Egr-1的3′UTR靶向结合和活性调控关系,用Egr-1-wt野生质粒、Egr-1-mut突变质粒和miR-let-7 c-3 p mimics或NC mimics共转染细胞,设置miR-let-7 c-3 p mimics+Egr-1-wt、miR-let-7 c-3 p mimics NC+Egr-1-wt、miR-let-7c-3p mimics+Egr-1-mut和miR-let-7c-3p mimics NC+Egr-1-mut组;通过细胞转染miR-let-7c-3p mimics和inhibitor调控miRNA表达,设置阴性对照序列(miR-let-7c-3p mimics NC和miR-let-7c-3p inhibitor NC),qRT-PCR验证调控效果;蛋白质印迹法检测miR-let-7c-3p对Egr-1的表达影响;流式细胞术检测miR-let-7c-3p对PMA诱导的K562细胞分化抗原的影响.结果经过PMA体外诱导后,与对照组比较,实验组K562细胞增殖水平升高(0.85±0.03 vs 0.46±0.03;t=16.050,P<0.001),CD11b阳性表达量升高〔(49.47±3.48)%vs(3.54±0.54)%;t=24.070,P=0.002〕,CD14阳性表达量也升高〔(59.84±5.26)%vs(6.79±0.66)%;t=16.670,P=0.004〕.与对照组比较,实验组Egr-1基因和蛋白表达水平升高,miR-let-7c-3p基因表达量降低,差异有统计学意义,均P<0.001.siRNA干扰实验结果显示,PMA+si-Egr-1组CD14分化抗原表达水平低于PMA+si-Ctrl组〔(7.03±1.45)%vs(24.40±4.70)%;t=6.113,P=0.004〕.双荧光素酶报告基因实验结果显示,miR-let-7c-3p mimics+Egr-1-wt组的荧光素酶活性低于miR-let-7c-3p mimics NC+Egr-1-wt组(0.77±0.006 vs 1.00±0.008;t=27.582,P<0.001).蛋白质印迹法检测结果显示,K562细胞转染miR-let-7c-3p inhibitor后,Egr-1蛋白表达水平高于miR-let-7c-3p inhibitor NC组(0.83±0.12 vs 0.39±0.00;t=-6.416,P=0.003);转染miR-let-7c-3p mimics后,Egr-1蛋白表达水平低于miR-let-7c-3p mim-ics NC组(0.18±0.01 vs 0.48±0.06;t=8.421,P=0.001).在miR-let-7c-3p对100μg/L PMA诱导K562细胞分化48 h的实验中,与0μg/L PMA组比较,100μg/L PMA可以诱导K562细胞分化抗原CD11b升高,分别为(3.44±0.43)%和(45.14±1.40)%,100μg/L PMA诱导K562细胞的同时转染miR-let-7c-3p inhibitor NC和miR-let-7c-3p in-hibitor后,2组分化抗原CD11b分别为(43.91±1.00)%和(59.22±1.28)%,4组间CD11b表达水平差异有统计学意义,F=1460.318,P<0.001.与0μg/L PMA组比较,100μg/L PMA同样诱导K562细胞的CD14表达升高,分别为(4.91±0.42)%和(70.54±1.45)%,100μg/L PMA共转染miR-let-7c-3p inhibitor NC或miR-let-7c-3p inhibitor后,CD14表达水平分别为(71.91±0.75)%和(85.98±0.52)%,4组间CD14表达水平差异有统计学意义,F=5163.346,P<0.001.结论miR-let-7c-3p可与Egr-1的3′UTR结合,并且可以靶向调控其表达水平变化.miR-let-7c-3p/Egr-1信号轴是白血病细胞定向分化为单核/巨噬细胞的重要途径之一.

Objective To investigate the role and molecular mechanism of miR-let-7 c-3 p/early growth response factor-1(Egr-1) signaling axis in leukemia cell-directed monocyte/macrophage differentiation.Methods Human chronic myeloid leukemia K562 cells were induced to differentiate into monocytes/macrophages with 100 μg/L phorbol 12-myristate 13-acetate(PMA,experimental group) and 0 μg/L PMA(control group) solution for 48 h, respectively.Differentiation antigens were determined by cytometry to confirm the successful establishment of the differentiation model.quantitative real-time polymerase chain reaction(qRT-PCR) was used to detect the expression changes of Egr-1 and miR-let-7 c-3 p before and after differentiation of leukemia cells, and Western blotting was used to detect the changes of Egr-1 protein expression.The effect of Egr-1 on cell differentiation was detected by siRNA interference experiment.PMA+si-Egr-1 group and PMA+si-Ctrl group were set up.The dual luciferase binding experiment was used to detect the 3′ UTR targeting binding and activity regulation of miR-let-7 c-3 p and Egr-1,cells were co-transfected with Egr-1-wt wild plasmid, Egr-1-mut mutant plasmid and miR-let-7 c-3 p mimics or NC mimics, setting miR-let-7 c-3 p mimics+Egr-1-wt, miR-let-7 c-3 p mimics NC+Egr-1-wt, miR-let-7 c-3 p mimics+Egr-1-mut and miR-let-7 c-3 p mimics NC+Egr-1-mut group.The miRNA expression was regulated by transfecting cells with miR-let-7 c-3 p mimics and inhibitor, and negative control sequences(miR-let-7 c-3 p mimics NC and miR-let-7 c-3 p inhibitor NC were set),qRT-PCR to verify the regulatory effect.Western blotting was used to detect the effect of miR-let-7 c-3 p on the expression of Egr-1.The flow cytometry was used to detect the effect of miR-let-7 c-3 p on PMA-induced K562 cell differentiation antigens.Results After in vitro induction by PMA,compared with the control group, the proliferation level of K562 cells in the experimental group increased(0.85±0.03 vs 0.46±0.03,t=16.050,P<0.001),and the positive expression of CD11 b increased [(49.47±3.48)% vs(3.54±0.54)%,t=24.070,P=0.002],and the positive expression of CD14 also increased [(59.84±5.26)% vs(6.79±0.66)%,t=16.670,P=0.004].Compared with the control group, the expression levels of Egr-1 gene and protein in the experimental group were increased, and the expression of miR-let-7 c-3 p gene was decreased, and the differences were statistically significant, both P<0.001.The results of siRNA interference experiment showed that the expression level of CD14 differentiation antigen in PMA+si-Egr-1 group was lower than that in PMA+si-Ctrl group [(7.03±1.45)% vs(24.40±4.70)%,t=6.113,P=0.004].The results of the dual luciferase reporter gene assay showed that the luciferase activity of co-transfected miR-let-7 c-3 p mimics and Egr-1-wt wild plasmid vector group was lower than that of co-transfected miR-let-7 c-3 p mimics NC and Egr-1-wt mutant plasmid vector group(0.77±0.06 vs 1.00±0.08,t=27.582,P<0.001).The results of western blotting showed that after K562 cells were transfected with miR-let-7 c-3 p inhibitor, the protein expression level of Egr-1 was higher than that in the miR-let-7 c-3 p inhibitor NC group(0.83±0.12 vs 0.39±0.00,t=-6.416,P=0.003).After transfection of miR-let-7 c-3 p mimics, Egr-1 protein expression level was lower than that in miR-let-7 c-3 p mimics NC group(0.18±0.01 vs 0.48±0.06,t=8.421,P=0.001).In the experiment of miR-let-7 c-3 p on 100 μg/L PMA to induce K562 cell differentiation for 48 h, compared with 0 μg/L PMA group, 100 μg/L PMA could induce the increase of K562 cell differentiation antigen CD11 b, which respectively was(3.44±0.43)% and(45.14±1.40)%.After K562 cells were induced by 100 μg/L PMA and transfected with miR-let-7 c-3 p inhibitor NC and miR-let-7 c-3 p inhibitor, the two groups of CD11 b were respectively(43.91±1.00)% and(59.22±1.28)%,F=1 460.318,P<0.001.Compared with the 0 μg/L PMA group, 100 μg/L PMA also induced an increase in the expression of CD14 in K562 cells by(4.91±0.42)% and(70.54±1.45)%,respectively.The 100 μg/L PMA co-transfected miR-let-7 c-3 p inhibitor NC or miR-let-7 c-3 p inhibitor, and CD14 expression levels were(71.91±0.75)% and(85.98±0.52)%,F=5 163.346,P<0.001.Conclusions miR-let-7 c-3 p can bind to the 3′UTR of Egr-1 and can target and regulate its expression level changes.The miR-let-7 c-3 p/Egr-1 signaling axis is one of the important pathways for the directed differentiation of leukemia cells into monocytes/macrophages.