miR-124靶向镁转运蛋白1调控活化后人T细胞功能耗竭

基础医学与临床 ›› 2019, Vol. 39 ›› Issue (10): 1404-1409.

miR-124靶向镁转运蛋白1调控活化后人T细胞功能耗竭

袁紫林,刁波

中国人民解放军中部战区总医院

收稿日期:2019-01-14 修回日期:2019-07-02 出版日期:2019-10-05 发布日期:2019-09-25
通讯作者: 刁波 E-mail:dpitao@163.com

miR-124 targets magnesium transporter 1 to regulate the function exhaustion of activated human T cells

Received:2019-01-14 Revised:2019-07-02 Online:2019-10-05 Published:2019-09-25

摘要/Abstract

摘要： 目的研究miR-124靶向镁转运蛋白1（MagT1）调控活化后T细胞功能耗竭。方法 1）分离外周血单个核细胞，用IFN-γ、IL-2、抗-CD3抗体、抗-CD28抗体和IL-1α活化T细胞；流式细胞仪检测T细胞活化标记分子CD25和CD69所占的比例。2）构建miR-124/ miR-124 sponge慢病毒载体；包装慢病毒后感染活化后T细胞，用RT-qPCR检测感染后T细胞内miR-124和MagT1基因表达。3）生物信息学分析miR-124与MagT1的靶向位点，并用双荧光素酶报告基因系统鉴定。4）Western blot法检测慢病毒感染前后T细胞MagT1和PD-1蛋白水平。5）CCK8法检测慢病毒感染前后T细胞增殖能力；ELISA法检测细胞T分泌细胞因子TNF-α和TGF-β的能力。结果流式细胞仪检测表明T细胞活化成功。RT-qPCR检测表明慢病毒构建成功，miR-124/ miR-124 sponge载体分别显著上调或下调miR-124的表达（P<0.01）。双荧光素酶报告基因系统证实miR-124靶向MagT1 3’UTR并抑制其表达。Western blot 表明miR-124过表达组MagT1蛋白水平低于对照组（P<0.05），PD-1蛋白水平高于对照组（P<0.05），抑制miR-124后，MagT1蛋白水平高于对照组（P<0.05），PD-1蛋白水平低于对照组（P<0.05）。miR-124过表达使T细胞增殖能力和分泌TNF-α能力显著降低，而抑制miR-124使T细胞增殖能力和分泌TGF-β能力显著升高（P<0.01）。结论miR-124可以负向调节靶基因MagT1的表达，并对活化后T细胞功能耗竭具有重要的调节作用。

关键词: 微小RNA-124, 镁转运蛋白1, T细胞耗竭

Abstract: Objective To investigate the regulation of miR-124 targeting magnesium transporter 1 on activated function exhaustion of activated T cell. Methods 1) Isolated monocytes from peripheral blood，then treated with IFN-γ, IL-2, anti-CD3 antibody , anti-CD28 antibody and IL-1α to activate T cell. The proportion of CD25 and CD69 subgroups was detected by Flow cytometry. 2) miR -124 / miR-124 sponge lentiviral vector was constructed. After lentivirus infected activated T cell, the expression of miR-124 and MagT1 gene in T cells was detected by RT-qPCR. 3) The targeted sites of miR-124 and MagT1 were analyzed by bioinformatics, and identified by dual luciferase reporter gene system.4) The expression of MagT1 protein and PD-1 protein were detected by Western blot. 5) The proliferation and secretion of TNF-αand TGF-β was detected by CCK8 and ELISA.Results Flow cytometry showed that T cells were activated.RT-qPCR indicated that the construction of lentivirus was successful, and miR-124/miR-124 sponge respectively up-regulated or down-regulated the expression of miR-124 (P<0.01).The dual luciferase reporter system confirmed that miR-124 targeted MagT1 3’UTR and inhibited its expression.Western blot showed that the MagT1 protein level in the miR-124 over-expression group was lower than that in the control group (P<0.05), and the pd-1 protein level was higher than that in the control group (P<0.05). After the inhibition of miR-124, the MagT1 protein level was higher than that in the control group (P<0.05), and the pd-1 protein level was lower than that in the control group (P<0.05). Over-expression of miR-124 significantly decreased the proliferation and secretion of TNF-α of T cells, while inhibiting of miR-124 expression significantly increased the proliferation and secretion of TGF-βof T cells (P<0.01).Conclusion It was confirmed that miR-124 could negatively regulate the expression of the targeted gene MagT1 and play an important regulatory role in the function exhaustion of activated T cells.

Key words: miR-124, magnesium transporter 1, T cell exhaustion

袁紫林刁波. miR-124靶向镁转运蛋白1调控活化后人T细胞功能耗竭[J]. 基础医学与临床, 2019, 39(10): 1404-1409.