上调miR-125a-5p抑制急性髓系白血病细胞系的增殖和侵袭

摘要/Abstract

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。

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

Abstract: 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 induce apoptosis by targeting at TRIM71. In addition, miR-125a-5p can inhibit the proliferation of AML cells by inhibiting the activation of the NF-κB signaling pathway.

Key words: acute myeloid leukemia, miR-125a-5p, proliferation, invasion, apoptosis, NF-κB signaling pathway

中图分类号:

R733.71

高秋英, 荀利如, 李岚, 侯丽敏, 周伟, 王晖. 上调miR-125a-5p抑制急性髓系白血病细胞系的增殖和侵袭[J]. 基础医学与临床, 2021, 41(11): 1594-1605.

GAO Qiu-ying, XUN Li-ru, LI Lan, HOU Li-min, ZHOU Wei, WANG Hui. Up-regulation of miR-125a-5p inhibits proliferation and invasion of acute myeloid leukemia cell lines[J]. Basic & Clinical Medicine, 2021, 41(11): 1594-1605.

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