Autoimmunity (Aug 2021)
MicroRNA-340-5p inhibits endothelial apoptosis, inflammatory response, and pro-coagulation by targeting KDM4C in anti-neutrophil cytoplasmic antibody (ANCA)-mediated glomerulonephritis through activation of B cells
Abstract
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, a class of systemic autoimmune diseases, results in damage of various critical organs including kidneys, lungs, eyes, and nervous system. MicroRNA-340-5p was confirmed to be downregulated in autoimmune pathogenesis. However, the role of miR-340-5p remains unknown in ANCA-induced glomerulonephritis (GN). The current study aimed to explore the role of miR-340-5p in ANCA-induced GN. The animal models of ANCA-induced GN was established by experimental autoimmune vasculitis (EAV) operation. The primary glomerular endothelial cells (PGEnCs) were treated with anti-myeloperoxidase (anti-MPO) to mimic cell injury in vitro. The renal function was analysed by measuring serum creatinine, blood urea nitrogen, urine blood, urine protein and urine leukocytes. The levels of RNA and proteins were examined by RT-qPCR and western blot analysis, respectively. The binding capacity between miR-340-5p and KDM4C was detected by luciferase reporter assay. Cell apoptosis was analysed by flow cytometry in vitro. Cell viability was determined by CCK-8 assay. The cleaved caspase-3 activity was analysed by immunofluorescent assay. Cell inflammation was measured by western blot. Cell procoagulant activity was assessed by FXa generation assay. The histological changes of renal tissues were assessed by Haematoxylin and eosin (H&E) staining assay. The correlation between miR-340-5p and KDM4C level (or content of TNF-α and IL-6) was analysed by Pearson correlation analysis. The injection of anti-MPO IgG induced a significant elevation of Serum creatinine and blood urea nitrogen in serum, as well as urine blood, urine protein and urine leukocytes. Importantly, KDM4C was downregulated in model group. In mechanism, we identified that miR-340-5p bound with KDM4C 3'untranslated region (UTR), negatively regulated KDM4C in endothelial cells and negatively correlated with KDM4C in serum of GN rats. In function, we found that miR-340-5p promoted B cell activation and proliferation by downregulating KDM4C. The in vitro assays showed that the decrease of cell viability induced by anti-MPO was reversed by miR-340-5p overexpression, and further reduced by KDM4C overexpression. Inversely, the suppressive effects of miR-340-5p mimics on cell apoptosis, cleaved caspase-3 activity, inflammatory response and pro-coagulation were countervailed by KDM4C overexpression in anti-MPO-treated cells. The in vivo assays validated that miR-340-5p overexpression mitigated the impairment of renal function, and histological changes induced by anti-MPO IgG injection in model group. Finally, we found the negative correlation between miR-340-5p and TNF-α (or IL-6) content in serum of GN rats. In conclusion, we found that miR-340-5p inhibited endothelial apoptosis and inflammatory response by targeting KDM4C in ANCA-mediated GN through activation of B cells, implying a potential novel insight for treatment of ANCA-mediated GN.
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