Smad1/5/8-mediated signaling [9]. However, the molec- ular mechanisms that regulate the cellular responses of cardiomyocytes to TGF-β signaling, which antagonizes cardiac hypertrophy, still have still not been elucidated. MicroRNAs (miRNAs) are small, endogenous non- coding RNAs, which are approximately 22 nucleotides in length. The miRNA inhibit the expression of specific genes by either degrading the target mRNA or direct translational inhibition [10]. Increasing evidences have revealed that miRNAs play important roles in cardiac hy- pertrophy and dysfunction [11-14]. Several studies have shown that miRNA expressions are altered in mouse and human HFs. The cardiac-specific knockout of Dgcr8 or Dicer, which are essential for the biogenesis and process- ing of miRNA, leads to a rapidly progressive dilated cardiomyopathy, HF and postnatal lethality [11-13]. The cardiac overexpression of miR-195, which is upregulated during cardiac hypertrophy, induces pathological cardiac growth and HF in transgenic mice [14]. Similarly, car- diac overexpression of miR-208a, which is located in the non-coding region of the α-myosin heavy chain (α-MHC) gene, is sufficient to induce hypertrophy, while analyses of miR-208a-deficient mice exhibited defects in stress- dependent cardiac growth and proper cardiac conduction [15, 16]. Previous studies have also shown that forced expression of miR-1 or miR-133 prevented hypertro- phic cell growth in cultured cardiomyocytes and cardiac hypertrophy in transgenic mice [17, 18]. However, the function of TGF-β-regulated miRNAs in cardiac hyper- trophy has not yet been reported. In this study, we performed miRNA northern blot to examine whether the expression patterns of miRNAs whose expression are changed in mouse models of car- diac hypertrophy [14, 17, 19] were altered in hypertro- phic Smad4-deficient hearts. We observed that miR-27b was significantly upregulated in Smad4 mutant hearts, and miR-27b could be inhibited by TGF-β1. We also observed that overexpression of miR-27b in primary cultured cardiomyocytes promoted hypertrophic cell growth by targeting the peroxisome proliferator-activated receptor-γ (PPAR-γ). Consequently, miR-27b transgenic mice developed dilated cardiomyopathy and cardiac dysfunction. In addition, in vivo administration of antag- omir-27b in a transverse aortic constriction (TAC) mouse model attenuated cardiac hypertrophy and dysfunction, suggesting miR-27b as a therapeutic target for HF. Results miR-27b is upregulated in Smad4-deficient hearts and downregulated by TGF-β1 Targeted deletion of Smad4 in cardiomyocytes induces www.cell-research.com | Cell Research Jian Wang et al. npg 517 cardiac hypertrophy and HF [6]. Our previous study has shown that the cluster of miR-23a/miR-27a/miR-24- 2 can be inhibited by TGF-β1 [20]. We hypothesized that the cluster of miR-23b/miR-27b/miR-24-1, which was frequently dysregulated in mouse models of cardiac hypertrophy [14, 19], could also be inhibited by TGF-β signaling. We first performed miRNA northern blot to examine the expression of these miRNAs, and found that miR-27b expression was significantly increased in Smad4 mutant hearts (Figure 1A); the result was also confirmed by real-time PCR (Supplementary information, Figure S1A). We further examined the expression of miR-23b and miR-24-1 using real-time PCR, and found that they were all upregulated in Smad4 mutants (Supplementary information, Figure S1B). The expression of miR-27b in a wide range of tissues was also shown by northern blot (Figure 1B). To confirm that miR-27b was regulated by TGF-β signaling, we treated neonatal cardiomyocytes with TGF-β1. Real-time PCR and northern blot results showed that pre-miR-27b and mature miR-27b were downregulated when treated with TGF-β1 (Figure 1C and 1D), implying that cardiac miR-27b was inhibited by TGF-β1. The efficient response of cardiomyocyte to TGF-β1 was confirmed by plasminogen activator inhibi- tor-1 (PAI-1) upregulation (Figure 1C and 1D). The ex- pressions of miR-23b and miR-24-1 were also inhibited by TGF-β1 (Supplementary information, Figure S1C). To examine whether miR-27b could be regulated by TGF-β1 at the transcriptional level, we used the 2 900- bp promoter of miR-27b, which contained a potential Smad-binding site (CAGACAT), to drive expression of the luciferase reporter gene in NIH3T3 cells. Treatment with TGF-β1 significantly inhibited the miR-27b pro- moter activity, whereas no effect was observed when the construct contained the promoter with a mutated Smad- binding site (Figure 1E). All these data corroborate the hypothesis that TGF-β1 regulated miR-27b transcription. miR-27b overexpression promotes hypertrophic growth of cardiomyocytes To investigate the potential function of miR-27b in cardiomyocytes, we performed gain-of-function and loss-of-function experiments by using cultured rat neo- natal cardiomyocytes. We designed two adenoviral vec- tors encoding sense (Ad-miR-27b) and antisense miR- 27b (Ad-anti-miR-27b), respectively. Real-time PCR and northern blot analysis showed efficient overexpression of the pre- and mature miR-27b in Ad-miR-27b-infected cardiomyocytes and efficient knockdown of endogenous mature miR-27b in cardiomyocytes with Ad-anti-miR- 27b (Figure 2A and 2B). The cardiomyocytes infected with adenoviruses for