miR-125b通过靶向B7-H4对再生障碍性贫血T细胞活化的影响

Effect of miR-125b on T Cell Activation in Aplastic Anemia by Targetting B7-H4

目的:探讨miR-125b在再生障碍性贫血(AA)患者T细胞活化中的作用及分子机制。方法:纳入山东省滨州医学院附属医院从2018年1月至2021年10月30例AA患者,15名健康个体作为健康对照(HC)组,分离外周血单个核细胞(PBMC),RT-q PCR检测PBMC中miR-125b、B7-H4 mRNA的水平。免疫磁珠分选AA患者和健康对照者naive T细胞和non-naive T细胞,检测miR-125b、B7-H4 mRNA的水平。转染慢病毒LV-NC inhibitor、LV-miR-125b inhibitor至细胞中,流式细胞术检测T细胞活化。双荧光素酶报告基因实验检测miR-125b和B7-H4之间的靶向关系。RT-q PCR和Western blot检测miR-125b、CD40L、ICOS、IL-10 mRNA和B7-H4蛋白的水平。结果:与HC组比较,AA患者PBMC中miR-125b表达上调(P<0.05),B7-H4 mRNA表达下调(P<0.05),CD4+CD69+T细胞和CD8+CD69+T细胞比例较高(P<0.05)。AA患者的naive T细胞和non-naive T细胞中miR-125b的表达均显著上调,B7-H4 mRNA表达下调(P<0.05),且non-naive T细胞较naive T细胞更明显(P<0.05)。与NC inhibitor组比较,miR-125b inhibitor组中miR-125b的表达显著降低(P<0.05),PBMC中CD4+和CD8+T细胞上CD69的表达水平显著降低(P<0.05),miR-125b inhibitor和B7-H4-3′UTR-WT共转染后荧光素酶活性显著升高(P<0.05)。与NC inhibitor组比较,miR-125b inhibitor组细胞中B7-H4 mRNA和蛋白水平显著升高(P<0.05)。与miR-125b inhibitor+shRNA组比较,miR-125b inhibitor+sh-B7-H4组CD4+和CD8+T细胞上的CD69表达水平显著升高(P<0.05),CD40L、ICOS和IL-10mRNA水平亦显著升高(P<0.05)。结论:miR-125b可能通过靶向B7-H4促进AA患者T细胞活化。

Objective:To investigate the effect of miR-125b on T cell activation in patients with aplastic anemia(AA)and its molecular mechanism.Methods:A total of 30 AA patients were enrolled in department of hematology,Binzhou Medical University Hospital from January 2018 to October 2021,as well as 15 healthy individuals as healthy control(HC)group.Peripheral blood mononuclear cells(PBMCs) were isolated,in which the levels of miR-125b and B7-H4 mRNA were detected by RT-qPCR.Immunomagnetic beads were used to separate naive T cells and non-naive T cells from AA patients and healthy people to detect the levels of miR-125b and B7-H4 mRNA.Lentivirus LV-NC inhibitor and LVmiR-125b inhibitor were transfected into cells,and T cell activation was detected by flow cytometry.The dual-luciferase reporter gene assay was used to detect the targetting relationship between miR-125b and B7-H4.RT-qPCR and Western blot were used to detect the levels of miR-125b,CD40L,ICOS,IL-10 mRNA and B7-H4 protein.Results:Compared with HC group,the expression of miR-125b was up-regulated but B7-H4 mRNA was down-regulated in PBMCs of AA patients(P<0.05),and the proportions of CD4+CD69+ T cells and CD8+CD69+ T cells in PBMCs of AA patients were higher(P<0.05).The expression of miR-125b was significantly up-regulated but B7-H4 mRNA was down-regulated in both naive T cells and non-naive T cells of AA patients(P<0.05),and non-naive T cells was more significant than naive T cells(P<0.05).Compared with NC inhibitor group,the expression of miR-125b was significantly decreased,the expression level of CD69 on CD4+ and CD8+ T cells in PBMCs was also significantly decreased,while the luciferase activity was significantly increased after co-transfection of miR-125b inhibitor and B7-H4-3′UTR-WT in the miR-125b inhibitor group(P<0.05).Compared with NC inhibitor group,the mRNA and protein levels of B7-H4 were significantly increased in the miR-125b inhibitor group(P<0.05).Compared with miR-125b inhibitor+shRNA group,the expression levels of CD69 on CD4+ and CD8+ T cells were significantly increased,and the levels of CD40L,ICOS and IL-10 mRNA were also significantly increased in the miR-125b inhibitor+sh-B7-H4 group(P<0.05).Conclusion:MiR-125b may promote T cell activation by targetting B7-H4 in AA patients.