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

XU Luyao; ZHANG Jie; LIN Xu

doi:10.3969/j.issn.1674-3806.2024.08.03

Abstract：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-1β and 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-1β and 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.