Figure 4: p53-dependent regulation of miR-29c by IGF2.

(a) Quantification of pri-miR-29c level in IGF2-treated fibroblasts using TagMan pri-miRNA assay. Data were normalized to U6 expression. (b,c) Fibroblasts were transfected with p53 overexpression or knockdown plasmids, and then expression levels of miR-29c (b) and VEGF (c) were determined by TagMan miRNA assay and western blot, respectively. (d) Three putative p53 binding sites in the promoter of miR-29c were identified by in silico prediction, and the enrichment of p53 in miR-29c promoter region was determined by ChIP. (e) Diagram illustrating the site-specific mutations introduced in the reporter plasmid for miR-29c promoter (Pgl3-hsa-miR29c-pro-BS3-WT) (upper panel). Lower panel showed the luciferase activity in fibroblasts transfected with p53 and wild type (WT) or mutated (M) miR-29c promoter. (f) Tagman miRNA assay and western blot analysis showing the expression of miR-29c and VEGF in p53 null fibroblasts upon IGF2 treatment, respectively. (g) VEGF expression in fibroblasts transfected with p53 or vector control in the presence or absence of IGF2. (h) Schematic diagram illustrating how IGF2 can induce fibroblasts to secrete VEGF via the mediation of miR-29c in a p53-dependent manner. Three biological replicates were performed for in vitro assays. Bars, s.d.; *P<0.05; **P<0.01; ***P<0.001 by Student’s t-test.