in the Smad3-deficient mice [22]. Studies on cardiomyo- cyte-specific Smad5 mutant mice have also suggested that Smad5 is required for cardiac homeostasis [23]. In this study, we observed that miR-27b was upregulated in Smad4-deficient hypertrophic heart tissues. We also ob- served that TGF-β1 significantly inhibited miR-27b at the transcriptional level. Furthermore, we provided the first direct evidence to prove that miR-27b overexpression is sufficient to induce cardiac hypertrophy both in vitro and in vivo. All these findings suggest that TGF-β signaling might protect cardiomyocytes from hypertrophic growth by downregulating miRNAs that cause cardiac hypertro- phy when overexpressed. This new finding has increased our knowledge on the complex molecular mechanisms of TGF-β in maintaining cardiac homeostasis. Recent studies have shown that miRNA mediates TGF-β signal- ing for modulating several biological processes [20, 24]. miR-23b cluster miRNAs, including miR-27b, have been shown to regulate liver stem cell differentiation by tar- geting Smads [25]. Our data suggest that miR-23b cluster miRNAs could be inhibited by TGF-β1. Whether there is a reciprocal feedback loop between TGF-β signaling and miR-23b/miR-27b/miR-24-1 cluster in cardiomyocytes needs to be further investigated. It is noteworthy that miR-27b is frequently upregu- lated in pressure-overloaded hypertrophic hearts [14, 17, 19]. Recent studies also demonstrated that miR-27b displayed an overt myocardial expression during heart development [26]. However, the miR-27b function in cardiac hypertrophy has not been elucidated yet. In the present study, we elucidated the in vivo physiological and pathological consequences of miR-27b in cardiac hyper- trophy using both gain-of-function and loss-of-function approaches. Most importantly, we performed curative experiment to demonstrate that miR-27b inhibition was indeed beneficial for pressure overload-induced cardiac hypertrophy and HF. Previous studies have revealed that a limited number of miRNAs may constitute new thera- peutic targets for ischemic heart diseases [27-30] as well as hypertension-induced HFs [31, 32]. Our study showed that in vivo knockdown of miR-27b significantly attenu- ated hypertrophic effects caused by pressure overload, providing a new potential therapeutic target for pressure overload heart diseases. In this study, we identified that PPAR-γ was the target of miR-27b and partially mediated cardiac hypertrophy that resulted form miR-27b overexpression. PPAR-γ is a well-known critical regulator during cardiac hypertro- phy. PPAR-γ agonists inhibit mechanical stress-induced hypertrophy of cultured neonatal rat ventricular cardio- myocytes [33]. Cardiac hypertrophy induced by the pres- sure overload in heterozygous PPAR-γ-deficient mice www.cell-research.com | Cell Research Jian Wang et al. npg 525 is slightly more pronounced than that in the wild-type controls [34]. Studies on cardiomyocyte-specific PPAR-γ knockout mice have shown that PPAR-γ suppresses cardiac growth and embryonic gene expression and pro- tects the heart from oxidative damage [35, 36]. Previous studies have shown that miR-27b regulates adipocyte differentiation and modulates inflammatory responses by targeting PPAR-γ [37, 38]. In our study, we observed that miR-27b targeted PPAR-γ in cardiomyocytes. Support- ively, PPAR-γ expression was significantly downregu- lated in hypertrophic hearts of miR-27b transgenic mice. This was further confirmed by the rescue experiment, which showed that overexpression of PPAR-γ decreased the hypertrophic growth caused by miR-27b overex- pression. As miR-27b might target hundreds of mRNAs simultaneously, our results demonstrate that PPAR-γ is an important, but possibly not the only, target gene re- sponsible for cardiac hypertrophy in miR-27b transgenic mice. In conclusion, our study identified that TGF-β- regulated miR-27b is a regulator of cardiac hypertrophy, suggesting that antagonizing miR-27b might be a new therapeutic entry point for cardiac diseases. Materials and Methods Mouse strains To obtain mice with targeted deletion of Smad4 gene in heart, homozygous mice for the floxed Smad4 allele (Smad4flox/flox, ref- erenced as Smad4Co/Co before) [39] were bred with α-MHC-Cre transgenic mice, in which the expression of Cre recombinase is driven by the α-MHC promoter [6]. The genotyping was per- formed as described [6]. Cloning and expression of miRNAs A 500 bp genomic fragment encompassing the miR-27b coding region was amplified by PCR and ligated into adenoviral vectors to generate Ad-miR-27b using 293A cells. PCR primers for miR-27b were as follows: miR-27b sense: 5′-TTTCTCGAGGAAGATGCT- CACCAGCCCTTTA-3′ and miR-27b antisense: 5′-TTTTCTA- GAGCATCATCTTGCCAGCGACT-3′. The inhibitor of miR-27b used for target analysis was synthesized by Shanghai GenePharma Co., Ltd. Isolation, culture and treatment of neonatal cardiomyocytes Neonatal rat ventricular myocytes were isolated from 2-day-old pups, infected and stained as described [40]. Generation of miR-27b transgenic mice A mouse genomic fragment flanking the miR-27b was ampli- fied using primers 5′-TCACATTGCCAGGGATTACCAC-3′ and 5′-TCAGCACGCTGTTTGCACTCTT-3′, then subcloned into a cardiac-specific expression plasmid containing the α-MHC promoter and human growth hormone (GH) poly (A) signal. To genotype the mouse, genomic DNA was isolated from mouse- tail biopsies and analyzed by PCR using specific primers: 5′-