Fig. 7.

E2f3 is a putative target of miR-351 in proliferating C2C12 myoblasts. A: predicted target site of miR-351 in the 3′-untranslated region (UTR) of mouse E2f3. An E2f3 3′-UTR mutant (E2f3 mut) was produced by introducing mutation in the seed sequence as indicated. B and C: luciferase assays were performed at day 2 after transfection of a mock control or luciferase reporters bearing intact or mutant E2f3 3′-UTR into myoblasts. To measure the effect of miR-351 on luciferase activity, the luciferase reporter fused to an intact E2f3 3′-UTR was cotransfected with either miRNA antisense or mimic and compared with that of scramble control (Ctrl). The results were expressed as firefly luciferase activity relative to the renilla luciferase expressed from a cotransfected plasmid and serving as a transfection control. D: endogenous E2f3 protein levels were determined by Western blot 2 days after transfection of miR-351 mimic or a scramble control (Ctrl) in myoblasts. Quantification of Western blot results was performed using scanning and ImageJ software. Results are expressed as integrated optical density normalized to TATA box binding protein (TBP) content; *P ≤ 0.04, #P ≤ 0.004. E: E2f3 mRNA expression was determined during in vivo muscle regeneration at 1, 3, 4, 7, and 21 days postinjury by qRT-PCR. Data are reported as fold change compared with uninjured baseline (B) muscle, 95% confidence intervals, 4–6 per mice/time point, and **P < 0.001.