Figure 3.
Figure 3. miR669q and miR669a prevent skeletal myogenesis in postnatal cardiac progenitors. (A) Schematic representation of miR669q within the Sgcb gene. Sequence homology between miR669a and miR669q encoded by the Sfmbt2 gene (intron 10) and by Sgcb (intron 1), respectively, is reported on the bottom. (B) miR669q expression in wt and Sgcb-null (KO) clones after 5 d of serum starvation (2% HS d5; left). Northern blot for miR669q expression in differentiated wt and three differentiated Sgcb-null clones (H4, G5, and G2) confirmed the absence of miR669q in Sgcb-null cardiac progenitors (top right). miR669q signals were normalized for U2 snRNA hybridization. (C) miR669q in situ hybridization on serial sections from adult normal (wt heart) and dystrophic Sgcb-null and Sgca-null hearts. (D) Northern blot analysis confirmed the absence of miR669q and miR669a down-regulation in Sgcb-null hearts. U2 snRNA hybridization was used for sample normalization. (E) MyoD expression analysis in the Sgcb-null cardiac clone transfected with scrambled miRNA, miR669a, and miR669q. Three independent experiments were performed in triplicates and statistically analyzed using Student’s t test. P < 0.05. (F) Western blot analysis for MyoD expression in Sgcb-null cardiac clone transfected with scrambled miRNA (H4 Ven KO) and miR669q. (G) Immunofluorescence analysis on Sgcb-null cardiac clone (H4 Ven KO) transfected with scrambled miRNA (left) and miR669q (right) 7 d after serum starvation (2% HS). (H) Luciferase (luc.) activity was measured in triplicates 24 and 48 h after COS-7 cell transfection with pre-miR669a/MyoD 3′UTR (top left) and pre-miR669a/Pax3 3′UTR (top right). Luciferase activity is indicated by black bars in scrambled transfected cells and by white bars in pre-miR669a/pre-miR669q–transfected cells. Mutagenesis experiments were performed on MyoD 3′UTR target sequence (MyoDm), and luciferase activity was measured in triplicates 24 h after transfection. Luciferase activity was relieved upon mutations of MyoD (gray bars) in both pre-miR669a (bottom left)– and pre-miR669q (bottom right)–transfected cells. Scrambled transfected cells were used as negative controls (ctrl). Error bars indicate standard deviation, and p-values are shown. (I) Schematic representation of the molecular mechanism responsible for the aberrant skeletal muscle differentiation in Sgcb-null cardiac progenitors. Active molecules are shown in green; no active molecules are shown in red. Error bars, except for H, show means ± SEM. Bars, 50 µm.

miR669q and miR669a prevent skeletal myogenesis in postnatal cardiac progenitors. (A) Schematic representation of miR669q within the Sgcb gene. Sequence homology between miR669a and miR669q encoded by the Sfmbt2 gene (intron 10) and by Sgcb (intron 1), respectively, is reported on the bottom. (B) miR669q expression in wt and Sgcb-null (KO) clones after 5 d of serum starvation (2% HS d5; left). Northern blot for miR669q expression in differentiated wt and three differentiated Sgcb-null clones (H4, G5, and G2) confirmed the absence of miR669q in Sgcb-null cardiac progenitors (top right). miR669q signals were normalized for U2 snRNA hybridization. (C) miR669q in situ hybridization on serial sections from adult normal (wt heart) and dystrophic Sgcb-null and Sgca-null hearts. (D) Northern blot analysis confirmed the absence of miR669q and miR669a down-regulation in Sgcb-null hearts. U2 snRNA hybridization was used for sample normalization. (E) MyoD expression analysis in the Sgcb-null cardiac clone transfected with scrambled miRNA, miR669a, and miR669q. Three independent experiments were performed in triplicates and statistically analyzed using Student’s t test. P < 0.05. (F) Western blot analysis for MyoD expression in Sgcb-null cardiac clone transfected with scrambled miRNA (H4 Ven KO) and miR669q. (G) Immunofluorescence analysis on Sgcb-null cardiac clone (H4 Ven KO) transfected with scrambled miRNA (left) and miR669q (right) 7 d after serum starvation (2% HS). (H) Luciferase (luc.) activity was measured in triplicates 24 and 48 h after COS-7 cell transfection with pre-miR669a/MyoD 3′UTR (top left) and pre-miR669a/Pax3 3′UTR (top right). Luciferase activity is indicated by black bars in scrambled transfected cells and by white bars in pre-miR669a/pre-miR669q–transfected cells. Mutagenesis experiments were performed on MyoD 3′UTR target sequence (MyoDm), and luciferase activity was measured in triplicates 24 h after transfection. Luciferase activity was relieved upon mutations of MyoD (gray bars) in both pre-miR669a (bottom left)– and pre-miR669q (bottom right)–transfected cells. Scrambled transfected cells were used as negative controls (ctrl). Error bars indicate standard deviation, and p-values are shown. (I) Schematic representation of the molecular mechanism responsible for the aberrant skeletal muscle differentiation in Sgcb-null cardiac progenitors. Active molecules are shown in green; no active molecules are shown in red. Error bars, except for H, show means ± SEM. Bars, 50 µm.

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