Regulation of Fli1 by miR-145. (a) Four possible miR-145 binding sites were identified in the Fli1 3' UTR. Evolutionary conservation across four mammalian species is shown. Seed regions are indicated by grey boxes. (b) Luciferase assays show that the predicted sites can mediate silencing by miR-145. Approximately 60-bp regions containing wild-type (WT) miR-145 binding sites in the Fli1 3' UTR were cloned into pMIR-REPORT vector (Applied Biosystems). Identical constructs with single base-pair mutations (Mut) were generated (mutated bases, C to G, are indicated in italics and bold in the sequences). HEK293 cells were co-transfected with pMIR-REPORT and either negative control dsRNA or a synthetic miR-145 dsRNA (Pre-miR-145) and luciferase activity assayed after 48 hours. Signals were normalized to the control groups. Error bars indicate standard error of the mean (n = 2, P < 0.05 for control versus Pre-miR-145 with all WT constructs). (c) Larger regions of the mouse and human Fli1 3' UTRs (704 and 1,288 bp, respectively) were cloned into luciferase reporter vectors and luciferase activity was assayed 24 hours after cotransfection with synthetic miR-30a or miR-145 in HEK293 cells. Four replicate experiments were performed and values shown are normalized to the empty (plasmid only) transfections (P < 0.001 for both constructs, comparing empty and miR-145 transfections). (d) Site-directed mutagenesis was applied to the 704-bp mouse Fli1 3' UTR fragment. Two single base mutations were introduced in each of the seed regions of predicted target sites 2 to 4. Constructs were cotransfected with either synthetic let-7f (control) or miR-145 and luciferase activity was assayed after 24 hours (n = 4). (e) Relative Fli1 protein levels in VAECs were measured 72 hours post-transfection with either Pre-miR-145 or a dsRNA control. Nuclear extracts were prepared and expression was assayed by western blotting followed by densitometric analysis. The membrane was re-probed with a lamin A/C antibody as a loading control. Error bars indicate standard error of the mean. (f) Fli1 mRNA levels in VAECs 72 hours post-transfection were determined using qRT-PCR and normalized to GAPDH. Error bars represent standard error of the mean (n = 4).