上调miR-125a-5p抑制急性髓系白血病细胞系的增殖和侵袭  

Up-regulation of miR-125a-5p inhibitsproliferation and invasion of acute myeloid leukemia cell lines

作　　者：高秋英 荀利如[2] 李岚 侯丽敏[1] 周伟[1] 王晖[1] GAO Qiu-ying;XUN Li-ru;LI Lan;HOU Li-min;ZHOU Wei;WANG Hui(Department of Hematology,Shanxi Provincial People's Hospital,Xi'an 710068,China;Nephrotic Hemodialysis Center, Shanxi Provincial People's Hospital, Xi'an 710068, China)

机构地区：[1]陕西省人民医院血液科,陕西西安710068 [2]陕西省人民医院肾病血透中心,陕西西安710068

出　　处：《基础医学与临床》2021年第11期1594-1605,共12页Basic and Clinical Medicine

基　　金：陕西省社会发展科技攻关项目(2015SF065)。

摘　　要：目的研究miR-125a-5p对急性髓系白血病(AML)细胞系增殖和侵袭的调控作用。方法RT-qPCR检测AML患者骨髓标本、AML细胞系(U937和HL60)及外周血单个核细胞(PBMC)中miR-125a-5p和TRIM71的表达。将U937和HL60细胞分为对照组(control)、miR-125a-5p模拟物组(miR-125a-5p mimic)、miRNA阴性对照组(miR-NC)、TRIM71过表达质粒组(pcDNA3.1-TRIM71)和对照质粒组(pcDNA3.1-NC)。采用Lipofectamine 2000将miR-125a-5p mimic、pcDNA3.1-TRIM71及阴性对照转染U937和HL60细胞。MTT法检测细胞增殖,基质胶侵袭试验测定细胞侵袭,流式细胞测量术检测细胞凋亡。RT-qPCR或Western blot检测TRIM71、Bax、Bcl-2、NF-κB p65的表达。通过荧光素酶报告基因验证miR-125a-5p和TRIM71的靶向调控关系。结果与对照或PBMC相比,miR-125a-5p在AML患者骨髓样本和AML细胞系中的表达水平明显降低(P<0.05)。与对照组相比,miR-125a-5p mimic组的U937和HL60细胞的增殖和侵袭能力明显降低,而细胞凋亡率明显升高(P<0.05)。与对照组相比,pcDNA3.1-TRIM71组的U937和HL60细胞的增殖和侵袭能力明显升高(P<0.05)。荧光素酶报告基因检测显示,miR-125a-5p mimic有效抑制了pGL3-TRIM71-WT的荧光素酶活性。与miR-125a-5p mimic组相比,(miR-125a-5p mimic+pcDNA3.1-TRIM71)组的细胞活力和细胞侵袭率显著增加,细胞凋亡率显著降低(P<0.05)。与miR-125a-5p mimic组相比,(miR-125a-5p mimic+pcDNA3.1-TRIM71)组的nucleus NF-κB p65蛋白表达水平显著增加(P<0.05)。结论上调miR-125a-5p通过靶向抑制TRIM71来降低AML细胞系的增殖和侵袭并诱导凋亡;miR-125a-5p可通过抑制NF-κB信号通路的活化来抑制AML细胞增殖。Objective To find the regulatory effect of miR-125a-5p on the growth and invasion of acute myeloid leukemia(AML)cell lines.Methods The expression of miR-125a-5p and TRIM71 in bone marrow specimens of AML patients,AML cell lines(U937 and HL60)and PBMC were examined by RT-qPCR.The U937 and HL60 cells were divided into control group(control),miR-125a-5p mimic group(miR-125a-5p mimic),miRNA negative control group(miR-NC),TRIM71 over-expression plasmid group(pcDNA3.1-TRIM71)and control plasmid group(pcDNA3.1-NC).U937 and HL60 cells were transfected with miR-125a-5p mimic,pcDNA3.1-TRIM71 and negative control using Lipofectamine 2000.Cell proliferation was measured by MTT assay;Cell invasion was measured by Matrigel invasion assay;And cell apoptosis was detected by flow cytometry.The expression of TRIM71,Bax,Bcl-2,NF-κB p65 was detected by RT-qPCR or Western blot.The targeted regulation relationship between miR-125a-5p and TRIM71 was verified by luciferase reporter gene assay.Results Compared with healthy controls or PBMC,miR-125a-5p expression levels in bone marrow samples of AML patients and AML cell lines were significantly reduced(P<0.05).The proliferation and invasion of U937 and HL60 cells in miR-125a-5p mimic group were significantly reduced,while the apoptosis rate was significantly increased(P<0.05).Compared with control group,the proliferation and invasion of U937 and HL60 cells in pcDNA3.1-TRIM71 group significantly were increased(P<0.05).Luciferase reporter gene assay detection showed that miR-125a-5p mimic effectively inhibited the luciferase activity of pGL3-TRIM71-WT.Compared with miR-125a-5p mimic group,the cell viability and cell invasion rate of(miR-125a-5p mimic+pcDNA3.1-TRIM71)group were increased significantly,while the apoptosis rate was decreased significantly(P<0.05).The expression level of nucleus NF-κB p65 protein in(miR-125a-5p mimic+pcDNA3.1-TRIM71)group was significantly increased(P<0.05).Conclusions Up-regulation of miR-125a-5p may inhibit proliferation and invasion of AML cell lines and

关 键 词：急性髓系白血病 miR-125a-5p 增殖 侵袭 凋亡 NF-ΚB信号通路 

分 类 号：R733.71[医药卫生—肿瘤]