npg Function of miR-27b in cardiac hypertrophy 518 Figure 1 miR-27b upregulated in the hearts of cardiomyocyte-specific Smad4-deficient mice was inhibited by TGF-β1. (A) Northern blot showed that miR-27b was greatly increased in Smad4Flox/Flox; α-MHC-Cre mutant hearts (Mut) compared with Smad4Flox/+; α-MHC-Cre controls (Con). A RNA probe against the small housekeeping RNA U6 (106 nt) was used as a loading control. Lower: quantitative analysis of fold change in expression of miRNAs. *P < 0.05 relative to control mice. (B) Northern blot showed that miR-27b was expressed in multiple tissues of a 1-month-old mouse. Lower: quantitative analysis of tissue profile for miR-27b. (C) Real-time PCR showed that pre-miR-27b was decreased in cardiomyocytes treated with TGF-β1 for 12 and 24 h, and the TGF-β1 target molecule, Pai-1, was significantly increased in cardiomyocytes treated with TGF-β1. (D) Northern blot revealed that miR-27b was significantly downregulated upon the treatment of TGF-β1 for 12 and 24 h, along with the upregulation of PAI-1 shown by western blot. (E) NIH-3T3 cells transfected with reporter vectors containing native or mutated miR-27b promoter were treated with or without TGF-β1 (5 ng/ml) for 24 h, and then harvested for luciferase assay. pGL-3 basic and pGL3-CAGA were used as negative and positive controls, respectively. *P < 0.05 relative to respective con- trols. 48 h were immunostained for α-actinin to study the mor- phological changes. Immunostaining revealed that car- diomyocytes treated with phenylephrine (PE) or infected with Ad-miR-27b were significantly larger than cardio- myocytes infected with adenoviruses encoding green fluorescent protein (Ad-GFP) (Figure 2C-2E). Hypertro- phy induced by miR-27b overexpression was comparable to that induced by adrenergic agonist PE (Figure 2D and Cell Research | Vol 22 No 3 | March 2012