npg Function of miR-27b in cardiac hypertrophy 522 old miR-27b transgenic mice by using M-mode echocar- diography (Figure 5A and Supplementary information, Table S1). We observed thinning of the LV walls (Figure 5B and 5C), an increase in the LV internal diameter in diastole and systole (Figure 5D and 5E) and an increase in the ratio of the LV mass (LVM) to the body weight (BW) (Figure 5F). In addition, the ejection fraction (EF) showed a declining trend (Figure 5G). Fractional short- ening (FS), an echocardiographic index of LV contractile function, was also decreased in the miR-27b transgenic mice (Figure 5H). Functional invasive hemodynamic measurements were performed to confirm the occurrence of echocardio- graphic alterations in miR-27b transgenic mice. The LV diastolic end pressure (LVEDP), measured after induc- ing anesthesia and LV catheterization, was found to be increased (Figure 5I). The mean arterial blood pressure (MAP) was also slightly increased (Figure 5J). How- ever, the heart rates and other hemodynamic variables of the control and transgenic mice were not different (Supplementary information, Table S2). All these results reflected the impairment of cardiac function in miR-27b transgenic mice. PPAR-γ is a direct target of miR-27b in cardiomyocytes To reveal the potential mechanism of miR-27b in cardiac hypertrophy, we screened the candidate miR- 27b target mRNAs that have been reported to be in- volved in cardiac hypertrophy using a bioinformatic approach, as detailed at TargetScan (http://genes.mit. edu/targetscan/). This analysis led to the identification of PPAR-γ, in which mRNA 3′-UTR regions comprise the seed sequences and flanking nucleotides matching miR-27b, which is highly conserved among the human, mouse, rat and dog species (Supplementary information, Figure S3A-S3C). To confirm whether PPAR-γ is a direct target of miR-27b, we performed luciferase reporter as- says in HEK293 cells. Cotransfection of miR-27b with the luciferase reporter gene linked to the wild-type 3′- UTR of PPAR-γ strongly inhibited the luciferase activity, while cotransfection with anti-miR-27b rescued this ef- fect (Figure 6A). However, no effect was observed with the construct harboring a mutant segment of PPAR-γ 3′- UTR (seed sequence 5′-ACUGUGA-3′ was mutated to 5-′AGAAUUC-3′). Moreover, cotransfection of miR-24 (control miRNA; not complementary to the 3′-UTR of PPAR-γ) with the wild-type PPAR-γ 3′-UTR construct did not alter the luciferase activity, indicating that miR- 27b specifically targeted PPAR-γ (Figure 6A). The RT- PCR and real-time PCR results showed that the miR-27b overexpression did not affect the transcriptional level of PPAR-γ in cardiomyocytes (Figure 6B). However, the infection of HEK293 cells and rat neonatal cardio- myocytes with Ad-miR-27b reduced the PPAR-γ protein levels, whereas knockdown of endogenous miR-27b in these cells increased the PPAR-γ protein levels (Figure 6C). We further measured the PPAR-γ expression level in Smad4 mutant heart tissues, which showed increased miR-27b levels, and observed that PPAR-γ was signifi- cantly decreased in Smad4 mutants (Figure 6D). Consis- tently, PPAR-γ levels were significantly reduced in miR- 27b transgenic hearts (Figure 6E). To test the effect of PPAR-γ inhibition on cardiomyo- cytes, we infected cardiomyocytes with adenovirus that interfere with of PPAR-γ (Ad-si-PPAR-γ; Supplementary information, Figure S4A-S4C). Knockdown of PPAR-γ in cardiomyocyes increased the cell size (Supplementary information, Figure S4D) and upregulated the expression of fetal genes (Supplementary information, Figure S4E), indicating that PPAR-γ interference induced hypertrophy of cardiomyocytes. Furthermore, we developed Ad-PPAR-γ to overex- press PPAR-γ in cardiomyocytes. Western blot results showed that PPAR-γ was successfully overexpressed in cardiomyocytes infected by Ad-PPAR-γ (Figure 6F). In addition, PPAR-γ overexpression could reduce the increased cell surface area caused by miR-27b overex- pression (Figure 6F). All these observations suggest that miR-27b acts as a negative regulator of PPAR-γ at post- transcriptional levels, and PPAR-γ mediates the cardiac hypertrophic effect of miR-27b. Inhibition of miR-27b attenuated cardiac hypertrophy and dysfunction in a TAC mouse model To test whether miR-27b inhibition was truly benefi- cial for cardiac diseases, we injected antagomir-27b into mice subjected to pressure overload of the LV by TAC and followed the disease for additional 3 weeks (Supple- mentary information, Figure S5A). First, we tested the effect of antagomir-27b in vivo and found that mature miR-27b was significantly downregulated in hearts by antagomir-27b administration (Supplementary informa- tion, Figure S5B). In addition, measurements of HW/BW, HW/TL, fetal genes and histological examination indi- cated that antagomir-27b administration did not affect the morphology of hearts in unstressed mice (Supplementary information, Figure S5C-S5F). Expression levels of miR-27b were increased in mice after the TAC surgery (Figure 7A). The TAC-induced miR-27b overexpression was effectively downregulated by antagomir-27b treat- ments as shown by real-time PCR (Figure 7B). In the myocardium, antagomir-27b treatment significantly re- versed changes in PPAR-γ expression caused by pressure overload (Figure 7C). TAC-operated mice treated with Cell Research | Vol 22 No 3 | March 2012