miR-155调控巨噬细胞极化并经外泌体介导足细胞损伤的作用机制研究

Study on the mechanism of action of miR-155 regulating the polarization of macrophages and mediating podocyte injury through exosomes

目的探讨miR-155调控巨噬细胞极化并经外泌体介导足细胞损伤的作用机制。方法选择巨噬细胞RAW264.7和肾小球足细胞MPC5进行实验。将miR-155mimics、miR-155mimicsNC、miR-155inhibitor和miR-155inhibitorNC慢病毒转染至RAW264.7细胞,构建miR-155过表达/低表达巨噬细胞系及其对照,分离巨噬细胞外泌体。采用流式细胞术检测各组巨噬细胞M1/M2表型比值。采用Transwell法将RAW264.7细胞与MPC5细胞进行共培养,分别于共培养12h、24h后采用实时荧光定量聚合酶链反应(RT-qPCR)检测巨噬细胞miR-155表达水平;采用Westernblot法检测巨噬细胞IL-6、IL-10表达水平,以及肾小球足细胞synaptoporin和nephrin表达水平;采用酶联免疫吸附试验(ELISA)检测肾小球足细胞IL-1β、IL-10表达水平;采用TUNEL染色法检测肾小球足细胞凋亡情况。结果成功构建miR-155过表达/低表达巨噬细胞系并分离其外泌体。miR-155过表达可诱导巨噬细胞极化为M1型,抑制miR-155可诱导巨噬细胞极化为M2型。在miR-155mimics慢病毒转染至巨噬细胞24h后,其miR-155表达水平显著增加(P<0.05),并上调IL-6的表达水平(P<0.05),抑制IL-10的表达水平(P<0.05),经Transwell共培养使足细胞synaptoporin、nephrin表达水平下降(P<0.05),IL-1β表达水平升高(P<0.05),并促进足细胞发生凋亡。而转染miR-155inhibitor慢病毒至巨噬细胞后所得结果趋势与miR-155mimics相反。RT-qPCR检测结果显示,miR-155mimics组巨噬细胞外泌体中miR-155水平显著升高(P<0.05),miR-155inhibitor组巨噬细胞外泌体中miR-155水平显著降低(P<0.05)。结论miR-155过表达可诱导巨噬细胞极化为M1型,并通过外泌体介导引发足细胞损伤,而下调miR-155表达则逆转这一作用。

Objective To explore the mechanism of action of micro ribonucleic acid-155(miR-155)regulating the polarization of macrophages and mediating podocyte injury through exosomes.Methods Macrophage RAW264.7 and glomerular podocyte MPC5 were selected for the experiments.The miR-155 mimics,miR-155 mimics negative con-trol(NC),miR-155 inhibitor and miR-155 inhibitor NC lentiviruses were transfected into RAW264.7 cells,and miR-155 overexpressing/underexpressing macrophage cell lines and their controls were constructed and the macrophage exosomes were isolated.Macrophage M1/M2 phenotype ratio in each group was detected by using flow cytometry.RAW264.7 cells were co-cultured with MPC5 cells by using Transwell method,and the miR-155 expression level of macrophages was detected by using real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)after 12 hours and 24 hours of co-culture,respectively,and the expression levels of interleukin(IL)-6 and IL-10 in macrophages,as well as the expression levels of synaptoporin and nephrin in glomerular podocytes were detected by using Western blot.The expression levels of IL-1βand IL-10 in glomerular podocytes were detected by using enzyme-linked immunosorbent assay(ELISA),and the apoptosis of glomerular podocytes was detected by using TUNEL staining method.Results The miR-155 over-expressing/underexpressing macrophage cell lines were successfully constructed and their exosomes were successfully isolated.Overexpression of miR-155 could induce the polarization of macrophages to M1 type,and inhibition of miR-155 could induce the polarization of macrophages to M2 type.After miR-155 mimics lentiviruses were transfected into macro-phages for 24 hours,their miR-155 expression levels were significantly increased(P<0.05),and the expression levels of IL-6 were up-regulated(P<0.05),and the expression levels of IL-10 were inhibited(P<0.05),and the co-culture by using Transwell resulted in decrease in the expression levels of synaptoprin and nephrin in podocytes(P<0.05),and increase in the expression level of IL-1βand facilitated podocyte apoptosis.However,the trend of the results obtained after transfection of miR-155 inhibitor lentiviruses into macrophages was opposite to that of miR-155 mimics.The results of RT-qPCR detection showed that the level of miR-155 in macrophage exosomes was significantly increased in the miR-155 mimics group(P<0.05),and the level of miR-155 in macrophage exosomes in the miR-155 inhibitor group was signifi-cantly decreased(P<0.05).Conclusion Overexpression of miR-155 can induce macrophages to polarize into M1 type and induce podocyte injury mediated by exosomes,while downregulation of miR-155 expression reverses this effect.