Col25a1 transcripts are regulated by miR-499 in skeletal muscles. (A) Sequence alignment show the target site of miR-208a, miR-208b and miR-499 in the 3′UTR of mouse Col25a1, as predicted by miRDB. This site is highly conserved between human, dog, rat and mouse. A mutation in the seed region is indicated. (B) The expression of miR-208, miR-499 and Myh7b was analyzed by RT-qPCR from E12.5, E14.5 WT limb buds and E18.5 WT hindlimb muscles. Data are means ± s.e.m of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs E12.5. Two to six embryos were analyzed in each experiment. (C) Juvenile satellite cells were isolated from WT 10 day old mice and expanded. The expression profile of miR-208 and miR-499 was analyzed by RT-qPCR in cells cultured in proliferation medium (PM), or 2 (DM2), 3 (DM3) or 4 (DM4) days in differentiation medium. Data are means ± s.e.m of three different experiments. *P < 0.05 vs PM. (D) Expanded WT myoblasts were transfected in differentiation medium with 10 nM miRNA mimics (miR-Control, miR-208b and/or miR-499). miR-208b, miR-499, Col25a1, Myh7 and Myh7b mRNA levels were quantified by RT-qPCR 48 h after transfections. The values are means ± s.e.m of three different experiments, each performed in duplicate. *P < 0.05; ***P < 0.001 vs miR-Ctrl. (E) miR-499 directly represses Col25a1 3′UTR in luciferase assays in 10T1/2 fibroblasts, in a specific manner, since a specific miScript Target Protector (target p.) for the miR-208b/miR-499 binding site on the 3′UTR of the Col25a1 gene rescued the inhibition. The repression is abolished by mutation of the miR-499 binding site in the Col25a1 3′UTR. The values are means ± s.e.m. from three to six independent experiments, each performed in duplicate. Values in graphs represent means ± s.e.m. ***P < 0.001 vs miR-Ctrl + target p. Ctrl.