Fig. 5.

miRNAs are associated with c-Myc and are important for c-Myc’s roles in the regulation of myoblast proliferation and differentiation and muscle fibre hypertrophy. a Results of the ChIP-qPCR validation of 14 c-Myc-bound miRNAs with different peak values between GM and DM. NC represents a genomic region without an identified c-Myc-binding peak. b Relative miRNA expression after c-Myc knockdown in primary myoblasts. c CCK-8 assay was performed to assess the effect of the CI-5 miRNAs on myoblast proliferation. SCR, scramble miRNA. d CCK-8 assay was performed to assess the effect of the CR-5 miRNAs on myoblast proliferation. SCR, scramble miRNA. e EdU staining after transfection of CI-5s and CR-5s. Bar, 50 μm. f Proliferation rate of myoblasts transfected with CI-5s and CR-5s. g Relative mRNA expression of the differentiation marker genes after transfection with the CI-5 miRNAs. h Relative mRNA expression of the differentiation marker genes after transfection with the CR-5 miRNAs. i MyHC staining of primary myoblasts 72 h after transfection with CI-5s and CR-5s. j Myotube area (%)72 h after transfection with CI-5s and CR-5s. k CCK-8 assay was performed to assess the effect of c-Myc and the CI-5 inhibitors on myoblast proliferation. l Primary myoblasts were co-transfected with mixtures of the indicated vectors and miRNA inhibitors, and the expression of the muscle differentiation marker genes was then analysed. m CCK-8 assay was performed to assess the effect of c-Myc and the CR-5 mimics on myoblast proliferation. n Primary myoblasts were co-transfected with mixtures of the indicated vectors and miRNA mimics, and the expression of the muscle differentiation marker genes was then analysed. o Chicken breast muscles were injected with CI-5, CR-5 or NC agomirs, and the expression of the muscle differentiation marker genes was then analysed. p H–E staining of breast muscle fibre cross sections from chickens injected with CI-5, CR-5 or NC agomirs. q Fibre diameter of chicken breast muscles injected with CI-5, CR-5 or NC agomirs. r H–E staining of breast muscle fibre cross sections from chickens injected with CI-5 antagomirs + pWPXL-GFP, NC antagomir + pWPXL-GFP and CI-5 antagomirs + pWPXL-c-Myc-Δ269–277. s Fibre diameter of chicken breast muscles injected with CI-5 antagomirs + pWPXL-GFP, NC antagomir + pWPXL-GFP and CI-5 antagomirs + pWPXL-c-Myc-Δ269–277. The results are shown as the mean ± sem of three independent experiments. In k, m, different letters a–d indicate values of each group at 4 d are significantly different at p < 0.05 by Duncan’s multiple range test. In l, n, s, different letters a–d above the bars indicate significant differences (p < 0.05) by Duncan’s multiple range test. In c, d, f–h, j, o, q, ANOVA followed by Dunnett’s test was used. In a–d, independent sample t-test was performed to determine the significant differences between the groups. *p < 0.05; **p < 0.01; ns, no significant difference