miR-27b controls vascular sprouting. (A) RNA was isolated from human microvascular endothelial cells grown in control medium (C, control), treated with VEGF (1-10 ng/mL) or VEGF + PEDF (10nM) and subjected to miRNA array analysis. A fragment of miRNA array shows the opposing effect of VEGF and PEDF on the expression of select miRNA (P < .05 by the 1-tailed Student t test). (B) Real-time RT-PCR was performed with primers for miR-27b (left panel) and miR-126 (right panel), with Let-7a as an internal control. (C) Northern blot was probed for indicated miRNA or U6 RNA to assess loading. The UV image is shown. (D) Whole-mount in situ hybridization for pri-miR-27b. The sense strand probe was used as a negative control (top panel). (E) Fertilized Tg(fli1a:EGFP)^y1 zebrafish eggs were injected with MOs against miR-27b (MO-27b), control MOs (MO-C), or left untreated (NT). The embryos were photographed at 24 hpf. (F) Mice with subcutaneous LLC tumor xenografts (5-7 mm in diameter) were treated with daily intratumoral injections of MO-C or MO-27b (12.5 mg/kg). After 5 days, the tumors were excised, sectioned, and stained for the endothelial marker CD31 (red) and the proliferation marker Ki67 (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Arrowheads indicate proliferating endothelial cells. (G-I) Experiments shown in panel F were subjected to morphometric analysis with MetaMorph software. *P < .03 (G) and P < .003 (H). No significant difference was noted in panel I, with P = .47 by the 1-tailed Student t test.