Fig 1: MiR-135b-5p and miR-499a-3p repressed MEF2C expression.(A) Predicted human MEF2C-encoded mRNA contains a 3'-UTR element that is partially complementary to the indicated miRNAs. Above: predicted target binding sites for miR-135b-5p and miR-499a-3p, respectively. Below: predicted duplex combination between the human MEF2C 3'-UTR-wt/mut and miR-135b-5p, MEF2C 3'-UTR-wt/mut and miR-499a-3p. (B) Luciferase activity of pmirGLO3-MEF2C-wt (WT) or pmirGLO-MEF2C-mut (MUT) in HEK293 cells after miR-135b-5p (left) and miR-499a-3p (right) transfections. Data are shown as means ± SD based on three independent experiments, **P < 0.01. (C) Predicted human FOXN3-encoded mRNA contains a 3'-UTR element that is partially complementary to the indicated miRNAs. Above: predicted target binding sites for miR-135b-5p and miR-499a-3p, respectively. Below: predicted duplex combination between the human FOXN3 3'-UTR-wt/mut and miR-135b-5p, FOXN3 3'-UTR-wt/mut and miR-499a-3p. (D) Luciferase activity of pmirGLO3-FOXN3-wt (WT) or pmirGLO-FOXN3-mut (MUT) in HEK293 cells after miR-135b-5p (left) and miR-499a-3p (right) transfections. Data are shown as means ± SD based on three independent experiments, *P < 0.05; **P < 0.01. (E) Predicted human ATP8A1-encoded mRNA contains a 3'-UTR element that is partially complementary to the indicated miRNAs. Above: predicted target binding sites for miR-135b-5p and miR-499a-3p, respectively. Below: predicted duplex combination between the human ATP8A1 3'-UTR-wt/mut and miR-135b-5p, ATP8A1 3'-UTR-wt/mut and miR-499a-3p. (F) Luciferase activity of pmirGLO3-ATP8A1-wt (WT) in HEK293 cells after miR-135b-5p (left) and miR-499a-3p (right) transfections. Data are shown as means ± SD based on three independent experiments. (G) Western blot analysis of MEF2C expression in HEK293 cells transfected with the miRNA mimics and inhibitors (left) and compiled data of MEF2C protein level from three independent experiments (right). Columns, mean; Bars, ± SD; *P < 0.05. The full-length gels are presented in Supplementary Fig. S6. (H) Compiled data of MEF2C mRNA level analysis from three independent experiments after transfection of miRNAs is shown. Columns, mean; Bars, ± SD.
Fig 2: MiR-135b-5p and miR-499a-3p promote endothelial cell proliferation and migration.(A) Western blot analysis of MEF2C expression in HUVEC cells transfected with the indicated miRNAs mimics. The full-length gels are presented in Supplementary Fig. S7. (B) Compiled data from three independent experiments after transfection of miRNAs is shown. Columns, mean; Bars, ± SD; ***P < 0.001. (C) Western blot analysis of MEF2C expression in HUVEC cells transfected with the indicated miRNAs and pcDNA3.1A-MEF2C.The full-length gels are presented in Supplementary Fig. S7. (D) Compiled data from three independent experiments after transfection of miRNAs is shown. Columns, mean; Bars, ± SD; ***P < 0.001. (E) Photoimages of EdU incorporation assay of HUVECs that were treated with miRNAs or rescue plasmid. Results are representative data from three independent experiments. (F) Proliferation rates of HUVECs that were treated with miRNAs or rescue plasmid. Results are presented as means ± SD of three independent experiments. *P < 0.05; **P < 0.01. (G) Photoimages of scratch assay of HUVECs that were treated with miRNAs or rescue plasmid. Results are representative data from three independent experiments. (H) Migration rates of HUVECs that were treated with miRNAs or rescue plasmid. Results are presented as means ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (I) Photoimages of transwell assay of HUVECs that were treated with miRNAs or rescue plasmid. Results are representative data from three independent experiments. (J) Migration rates of HUVECs that were treated with miRNAs or rescue plasmid. Results are presented as means ± SD of three independent experiments. *P < 0.05; **P < 0.01.
Fig 3: Putative mechanism by which miR-135b-5p and miR-499a-3p might target MEF2C to promote cell proliferation and migration.See the discussion section for further details.
Fig 4: A, B) Changes of mRNA level of 84 genes involved in glucose metabolism (A) or DNA transcription (B) between podocytes cultured in normal glucose (NG) or HG as analyzed by quantitative PCR microarray. Tables in panels show genes whose expression levels were statistically different between both groups. A scheme of the main regulatory network identified in podocyte remodeling by hyperglycemia. C–F) Complex IV (C), PGC-1a (D), and podocin (E) expression of podocytes cultured with esiRNA of EGFP, MYF5 (F), MEF2C, and MYF5 + MEF2C were determined by Western blotting.
Fig 5: Representative images of immunostaining of kidney-biopsied specimens from normal participants and patients with diabetic nephropathy. Arrows indicate positive podocytes. Percentages of positive cells for PGC-1a, MYF5, and MEF2 per total cells in glomeruli were calculated. For pyruvate kinase, numbers of positive podocytes per glomerulus were counted. *P < 0.05, **P < 0.01, unpaired Student's t test.
Supplier Page from Abcam for Anti-MEF2C antibody [EPR1452(N)]