Fig. 4

RCN3 was a direct target of miR-92a-1-5p and exosomal miR-92a-1-5p derived from HK-2 cells promoted MFT through suppressing RCN3 in MMCs. A RCN3 expression in MMCs after transfection with miR-92a-1-5p mimic (100 nM) or mimic control (miR-NC, 100 nM). B HEK 293 cells were co-transfected with pGL3-RCN3-3′UTR luciferase plasmid/pRL-TK Renilla (8:1) or pGL3- RCN3-3′UTR MT luciferase plasmid/pRL-TK Renilla (8:1) with various miRNA mimics (control mimic or miR-92a-1-5p mimic) using DharmaFECT Duo Transfection Reagent. After 48 h, both firefly and Renilla luciferase activities were quantified using the Dual-Glo® Luciferase Assay System. C, D RCN3 mRNA and protein expression in MMCs treated with HG-treated HK-2 cell-derived exosomes for 48 h. E After transfection with miR-92a-1-5p inhibitor (50 nM) or control inhibitor (anti-miR-NC, 50 nM) for 24 h, MMCs were incubated with exosomes derived from HK-2 cells under NG or HG condition. Western blotting was used to measure RCN3 protein expression. F The level of RCN3 in MCs of kidneys in mice and humans. The kidney sections of non-diabetic db/m mice (n = 4) and diabetic db/db mice (n = 4) at 12th week, and human donors (UTUC with normal kidney function and normal glomeruli) (n = 4) and patients with DN (n = 4) were co-stained with RCN3 (brown) and α-SMA (green), as the marker of MCs. Arrow shows as MCs. G MMCs were transfected with RCN3 siRNA, and 24 h after transfection, the cells were treated with NG for 48 h. H, I MMCs were transfected with RCN3 cDNA or control plasmid, and 24 h after transfection, the cells were treated with HG-treated HK-2 cell-derived exosomes or transfected with miR-92a-1-5p for 48 h. N-cadherin, vimentin and E-cadherin expressions were assessed using western blotting The images quantification was performed using the IHC Profiler Plugin of ImageJ Software. The bar graph represents the mean ± S.E.M. of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test or ANOVA followed by the post hoc test with Tukey’s correction