Fig 1: Effect of the transport protein HNRNPA1 on the exosomal sorting of miR-483-5p in renal TECs.A The primary mouse renal TECs were cultured under NG and HG conditions for 48 h, and their exosomes were isolated for verification by TEM (Scale Bar = 100 nm) and B western blot (CD63 and CD9 proteins). C Detection of the miR-483-5p levels in cells and exosomes by qRT-PCR. D After HG treatment for 48 h, the RNA pull-down of miR-483-5p was carried out, and the eluate was subjected to mass spectrometry. E Gene Ontology (GO) analysis. F RNA immunoprecipitation verification of HNRNPA1 and HNRNPA2/B1 that bound to miR-483-5p. G RNA pull-down experiment corroborated the binding of HNRNPA1 to miR-483-5p. H After transfecting si-hnrnpa1 into primary mouse renal TECs, the cells were treated with 30 mM HG for 48 h. Detection of the miR-483-5p levels in cells and exosomes by qRT-PCR. I After transfecting si-hnrnpa1 and/or miR-483-5p inhibitor into primary mouse renal TECs, the cells were treated with 30 mM HG for 48 h. Detection of HNRNPA1, CoL I, CoL III, TIMP2, ERK1/2, and pERK1/2 protein levels by western blot. **P < 0.01 vs. NG, IgG or HG + si-NC. ##P < 0.01 vs. Anti-HNRNPA1. Student’s t-test and one-way ANOVA followed by Tukey’s post-test. of three independent experiments. NG normal glucose, HG high glucose, NC negative control.
Fig 2: Effect of TIMP2 and MAPK1 on the regulation of miR-483-5p on renal TECs under HG conditions.A TargetScan and miRanda databases were applied to predict target genes that were efficiently bound to miR-483-5p both in mice and humans. B The top 10 genes (PTMA, SCRT1, CXXC5, TIMP2, CTDSPL2, NXF1, MAPK1, REM2, STK40, and CAMK1D) with strong conservative software prediction scores were applied to conduct the dual-luciferase reporter gene assay. C The binding sites of miR-483-5p and TIMP2, miR-483-5p and MAPK1 in mice and humans. D After transfecting si-TIMP2, si-MAPK1 into TCMK-1 cells, the cells were treated with 30 mM HG for 48 h. Detection of CoL I, CoL III, α-SMA, E-cadherin, Vimentin, Smad3, and nSnail protein levels by western blot. E After transfecting miR-483-5p mimic into TCMK-1 cells, the cells were treated with HG. Detection of TIMP2, ERK1/2 (MAPK1 encoding protein), and pERK1/2 protein levels by western blot. F RNA pull-down experiments corroborated that miR-483-5p bound to the 3′UTR region of TIMP2 and MAPK1, respectively. *P < 0.05 vs. HG or control. **P < 0.01 vs. HG, control or Bio-miR-ctrl. Student’s t-test. of three independent experiments. nSnail Snail in the nucleus, NC negative control, HG high glucose, ctrl control.
Fig 3: Transport mechanism.A Mechanism flow diagram. Under physiological conditions (non diabetic), most miR-483 (miR-483-5p) remained in the renal tubular epithelial cells, and only a small amount of miR-483 was encapsulated into MVB (multivesicular body, vesicles) and was secreted into the extracellular through exosomes. Therefore, the intracellular miR-483 blocked ECM synthesis by restraining the TIMP2/MAPK1 pathway; Under pathological conditions, most miR-483 was encapsulated into MVB and was secreted into the extracellular domain through exosomes and transferred into the urine. Therefore, miR-483 was highly expressed in the urinary exosomes, and the lack of miR-483 in renal tubular epithelial cells led to the activation of the TIMP2/MAPK1 pathway, thus promoting ECM synthesis. B Pearson correlation coefficient analysis of the correlation between urinary ACR and urinary exosome miR-483-5p. Pearson correlation coefficient analysis of three independent experiments.
Fig 4: Regulation of miR-483-5p on the renal interstitial fibrosis in type 1 and type 2 diabetic mice. A–D Detection of serum creatinine and urinary albumin to creatinine ratio concentrations by Creatinine Serum Detection Kit and urinary albumin to creatinine ratio assay kit. E–H Detection of TIMP2, ERK1/2, and miR-483-5p expressions by qRT-PCR and western blot. *P < 0.05, **P < 0.01 vs. control or db/m. #P < 0.05, ##P < 0.01 vs. STZ + AAV-control or db/db + AAV-control. One-way ANOVA followed by Tukey’s or LSD post-test. STZ Streptozotocin, db/m db/misty, ACR albumin to creatinine ratio.
Fig 5: miR-122-5p/TIMP2 regulates progress of ccRCC cells. (a) qRT-PCR detected the expression of TIMP2. (b) Western blot detected the expression of TIMP2 protein. (c) Colony formation assay detected the number of colonies. (d) EdU assay detected the EdU-positive cells. (e and f) MTT detected the cell viability. (g) Western blot detected the level of PCNA protein. (h and i) Transwell assay detected the cell migration and invasion ability. (j and k) Western blot detected the level of Twist1 and E-cadherin. (l) Cell apoptosis was detected by flow cytometry. *P < 0.05.
Supplier Page from Abcam for Anti-TIMP2 antibody [F27P3A4]