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

作　　者：齐福 王希娣 王朝喆 孙瑞婧 都鹏超 王静 姜国胜 QI Fu;WANG Xi-di;WANG Chao-zhe;SUN Rui-jing;DU Peng-chao;WANG Jing;JIANG Guo-sheng(Department of Immunology,School of Basic Medicine,Binzhou Medical College,Yantai 264003,China;Precision Medicine Laboratory,Jinan Zhangqiu District People’s Hospital,Affiliated to Jining Medical College,Jinan 250200,China;Immunotherapy Center,Shandong Yinfeng Institute of Life Sciences,Jinan 250102,China)

机构地区：[1]滨州医学院基础医学院免疫学教研室,山东烟台264003 [2]济宁医学院附属济南市章丘区人民医院精准医疗实验室,山东济南250200 [3]山东银丰生命科学研究院免疫治疗中心,山东济南250102

出　　处：《中华肿瘤防治杂志》2022年第8期554-563,共10页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81573467);山东省自然科学基金青年项目(ZR2020QH160);山东省自然科学基金面上项目(ZR2021MH080);济宁医学院教师科研扶持基金(JYFC2019FKJ102);济南市卫健委科技计划项目(2019-1-66)。

摘　　要：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 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 o

关 键 词：K562细胞 细胞分化 miR-let-7c-3p 早期生长反应因子-1 巨噬细胞 

分 类 号：R733.7[医药卫生—肿瘤]