FIGURE 6.

miR-9 inhibits myocardin expression. A, the miR-9 targeting sites in myocardin 3′-UTR are evolutionarily conserved in human, rat, and mouse. B, Iso induces a reduction of miR-9 levels. Cardiomyocytes were treated with 10 μm Iso. The cells were harvested at the indicated time for the analysis of miR-9 levels. *, p < 0.05 versus control. C, Aldo induces a reduction of miR-9 levels. Cardiomyocytes were treated with 1 μm Aldo. The cells were harvested at the indicated time for the analysis of miR-9 levels. *, p < 0.05 versus control. D, miR-9 suppresses myocardin translation. HEK293 cells were transfected with the luciferase constructs of the wild type myocardin-3′-UTR (Myocardin-3′-UTR-wt) or the mutated myocardin-3′-UTR (Myocardin-3′-UTR-mut), along with the expression plasmid for miR-9, miR-9 antagomir, or the antagomir negative control (Antagomir-NC). *, p < 0.05 versus myocardin-3′-UTR-wt; #, p < 0.05 versus myocardin-3′-UTR-wt plus miR-9. E, miR-9 suppresses the expression of myocardin in the cellular model. Cardiomyocytes were infected with adenoviral miR-9 or the mutated miR-9 (miR-9-mut) at the indicated moi. Myocardin expression was analyzed by immunoblot 48 h after infection. F, miR-9 suppresses the expression of myocardin in the animal model. Adult male C57BL/6 mice (8 weeks old) were infused with miR-9 mimic or the mimic negative control (mimic-NC) (30 mg/kg) as described under “Experimental Procedures.” Myocardin expression levels were analyzed by immunoblot 3 days after infusion. The data are expressed as the means ± S.E. of three independent experiments.