Fig. 5

miR-25 downregulated PGC-1beta protein expression by directly targeting its 3′-UTR. (a) Schematic illustration of the predicted binding sites of miR-25 in the 3′-UTR of PGC-1beta. Sequence conservation of three predicted miR-25 binding sites within the 3′-UTR of PGC-1beta in four ruminates (Capra hircus, Ovisaries, Bostaurus and bison). WT1 and WT2 represent, respectively, PGC-1beta 3′-UTR-1 (748bp) and PGC-1beta 3′-UTR-2(504bp). The wild type (WT) miRNA-binding sites (black) were modified to complementary sequences (red) to construct the mutated PGC-1beta 3′-UTR (Mut). Mut a, b, c and ab corresponds to mutated PGC-1beta 3′-UTR in site 1, 2, 3 and double mutation of sites 1 and 2, respectively. (b-c) Luciferase reporter assay to assess interaction between miR-25 and PGC-1beta 3′-UTR-1 (b) and PGC-1beta 3′-UTR-2 (c) in GMECs. Data represent the mean ± S.D. of three independent cell samples. *P < 0.05, **P < 0.01, ***P < 0.001