miR-27 regulates the expression of semaphorin 6A and 3B. (A) Expression of SEMA6A and SEMA3B in HUVECs after transfection with miR-27 precursor (pre-miR-27b, 1nM) after 48 and 72 hours. Representative Western blots are shown. Tubulin serves as loading control. Blots were scanned, and protein expression was quantified by densitometric analysis. The ratio of SEMA6A/tubulin or SEMA3B/tubulin is shown as fold change ± SEM; n = 3. (B) Expression of SEMA6A and SEMA3B in HUVECs after transfection with LNA-miR-27 inhibitor (0.1nM) after 48 and 72 hours. Representative Western blots are shown. Tubulin serves as loading control. The ratio of SEMA6A/tubulin or SEMA3B/tubulin is shown as fold change ± SEM; n = 3. (C) Expression of SEMA6A and SEMA3B in hearts of mice treated with antagomir-27 or antagomir-control (see Figure 3 for details). Representative Western blots are shown. ERK1 serves as loading control. The ratio of SEMA6A/ERK1 or SEMA3B/ERK1 is shown as percentage of control; n = 4. P < .05. (D) Luciferase normalized to Renilla activity was measured in homogenates of HEK293FT cells transfected with luciferase constructs containing the wild-type (wt) or mutated (mut) seed sequences of miR-27 together with pre-miR-27a, pre-miR-27b, or control pre-miR. Measurements were done 48 hours after transfection; n = 6 or 7. (E-F) HUVECs were transfected with siRNA (67nM) targeting SEMA6A. A siRNA directed against firefly luciferase was used as a control. (E) Expression of SEMA6A was measured by quantitative RT-PCR after 24 hours; n = 3. *P < .05. (F) Effect of SEMA6A silencing on endothelial cell sprouting; n = 4. P < .05 (paired t test). (G) HUVECs were incubated with 300 or 500 ng/mL human recombinant SEMA6A or SEMA3B protein, and endothelial cell sprout formation was measured; n = 3 or 4. *P < .05 versus control.