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. 2019 Jan 30;10(2):391–410. doi: 10.1002/jcsm.12374

Figure 5.

Figure 5

MiR‐15a, miR‐15b‐5p, and miR‐15c‐5p inhibit myoblast proliferation and differentiation. (A) RNA‐seq analysis found that miR‐15a, miR‐15b‐5p, and miR‐15b‐5p were down‐regulated in hypertrophic broilers compared with leaner broilers, and this expression pattern was validated by qRT‐PCR (B). (C) Cell cycle analysis of myoblasts at 48 h after transfection of miR‐15a, miR‐15b‐5p, miR‐15c‐5p, and miR‐NC. (D) CCK‐8 assay was performed to assess the effect of miR‐15a, miR‐15b‐5p, or miR‐15c‐5p overexpression on myoblast proliferation. (E) EdU and Hoechst (nuclei) staining analysis after transfection of miR‐15a, miR‐15b‐5p, or miR‐15c‐5p in proliferating myoblast, scale bars are 50 μm. (F) The proliferation rate of myoblast cells transfected with miR‐15a, miR‐15b‐5p, or miR‐15c‐5p. (G) Myoblast cells transfected with miR‐15a, miR‐15b‐5p, or miR‐15c‐5p were induced to differentiate for 72 hr then stained with MyHC antibody and DAPI (nuclei). Scale bars are 100 μm. (H) Myotube area (%) at 72 h after transfection of miR‐15a, miR‐15b‐5p, or miR‐15c‐5p. Overexpression of miR‐15a, miR‐15b‐5p, or miR‐15c‐5p decreased the RNA expression level of IGF‐1 pathway associated genes, including IGF1 (I), IGF1R (J), and IGF2R (K). Overexpression of miR‐15a, miR‐15b‐5p, or miR‐15c‐5p decreased the RNA expression level of myoblast cell differentiation associated genes, including MyoD (L), MyoG (M), MyHC (N), and MyoMarker (O). Results are shown as the mean ± SEM of three independent experiments. Independent sample t‐test was used to analysis the statistical differences between groups. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.