miR-124靶向镁转运蛋白1调控活化后人T细胞功能耗竭

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

目的研究miR-124靶向镁转运蛋白1(MagT1)可否调控活化后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。

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 luciferasereporter gene system.4) The expression of MagT1 protein and PD-1 protein were detected by Western blot. 5)The proliferation and secretion of TNF-α and TGF-β were 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 inhibited miR-124 expression in turn increased the proliferation and secretion of TGF-βof T cells(P<0.01). Conclusions miR-124 may negatively regulate the expression of the targeted gene MagT1 and regulate the function exhaustion of activated T cells.