Figure 1: miR-431 is predominantly expressed in the skeletal muscle and targets Pax7 in myogenic cells.

(a) miR-431 target sequence alignment in the Pax7 3′-UTR predicted with TargetScan. Two predicted target sites of miR-431 in the 3′-UTR of mouse Pax7 are shown. The nucleotide coordinate of Pax7 was based on the mouse Refseq (NM_011039). m1utr and m2utr, mutated from the seed matches, are indicated. (b) Expression of miR-431 in tissues from 3-week-old mice was analysed using northern blot analysis. Total RNA (20 μg) was loaded to detect miR-431, and tRNA was used as a loading control. (c,d) Expression of miR-431 and Pax7 during satellite cell proliferation (growth medium; GM) and differentiation (differentiation medium; DM) was assayed using real-time RT–qPCR. U6 small nuclear RNA (snRNA) served as an internal control for miR-431 expression, and GAPDH as a control for Pax7 detection. exp, expression. (e) Effect of miR-431 transfection in HEK 293 cells, measured with luciferase assays. Empty pGL-3 vector was a negative control. w-UTR is a Pax7-3′-UTR, with miR-431-binding site inserted downstream of the luciferase gene in the pGL-3 control vector. m1utr and m2utr are mutant forms of the Pax7 3′-UTR. A Renilla luciferase plasmid was co-transfected with a firefly luciferase construct as a transfection control. The results are expressed as firefly luciferase activity relative to Renilla luciferase activity. Values are means±s.e.m. of three independent measurements. **P<0.01. (f) miR-431 was overexpressed in C2C12 cells with a lentiviral system. Cells stably expressing a scramble sequences served as negative controls (NC). Pax7 expression was determined using RT–qPCR. Error bars represent means±s.e.m. of three independent experiments. ***P<0.001. Two-tailed Student’s t-test was used for all statistical testings.