npg Function of miR-27b in cardiac hypertrophy 520 Ki67 immunofluorescence showed that cardiomyocyte proliferation was not affected by miR-27b overexpres- sion (Supplementary information, Figure S2A). These findings indicate that miR-27b could induce hypertrophy of cardiomyocytes. Cardiomyocyte-specific overexpression of miR-27b in- duces cardiac hypertrophy To investigate the role of miR-27b in cardiac remod- eling in vivo, we generated two transgenic mouse lines carrying the mouse miR-27b DNA under the control of α-MHC promoter. Northern blot results revealed that miR-27b was successfully overexpressed in the trans- genic hearts from lines #1 and #2 (3-4-fold increase, Figure 3A). We selected line #1 for further studies. All these miR-27b transgenic mice were born normally and had normal cardiac structure (data not shown). They survived to adulthood and were fertile. However, 30% of transgenic mice died suddenly between 6 and 12 months of age. The miR-27b transgenic mice developed signifi- cant cardiac hypertrophy at 3 months of age (Figure 3B). They showed higher heart weight/body weight (HW/ BW) and heart weight/tibia length (HW/TL) ratios than the littermate controls (Figure 3C). The fetal genes such Figure 3 Overexpression of miR-27b induced cardiac hypertro- phy in vivo. (A) Northern blots and quantification data (n = 3) showed an approximately 3-4-fold increase of miR-27b expres- sion in hearts of miR-27b transgenic (Tg) mice compared with control (Con) littermates. A RNA probe against U6 was used as a loading control. (B) Gross morphology of miR-27b Tg hearts was enlarged compared with control hearts at 3 months. Scale bar, 5 mm. (C) Heart weight to body weight or tibia length of 3-month-old miR-27b Tg mice (n = 7) was significantly higher than controls (n = 8). (D) Analysis of ANF, β-MHC and SKA by real-time PCR in hearts from miR-27b Tg and control mice (n = 6/genotype). *P < 0.05 vs control mice. Figure 4 Overexpression of miR-27b in vivo induced cardiac hy- pertrophy. (A, B) Macroscopic view of H&E-stained transverse sections from control (A) and miR-27b Tg hearts (B). (C, D) Im- munostaining with a rabbit polyclonal anti-laminin antibody was performed on transverse sections of myocardium from littermate control (C) and miR-27b Tg mice (D). (E, F) Representative im- ages of isolated cardiomyocytes from 3-month-old littermate control (E) and miR-27b Tg mice (F). (G) Histological sections were detected by laminin staining to determine cell size. Mean cardiomyocyte cross-sectional area of miR-27b Tg hearts (n = 500) was significantly larger than control cardiomyocytes (n = 500). *P < 0.001. (H) The mean cell area of the cardiomyocytes (n = 150) isolated from 3-month-old miR-27b transgenic mice was greater than control cardiomyocytes (n = 150; *P < 0.001). Scale bars, A, B: 2 mm and C-F: 50 µm. as ANF, β-MHC and SKA were also significantly up- regulated in the heart tissues of miR-27b transgenic mice (Figure 3D). We performed further studies to ascertain whether the increase of the heart size was due to cell growth (increase in cell size) or cell proliferation (increase in cell num- ber). Cardiomyocyte proliferation was assessed by BrdU staining. The cardiomyocyte proliferation in the miR-27b transgenic mice and the littermate controls were not sig- nificantly different (Supplementary information, Figure S2B). Cross-sections of hearts confirmed left ventricle Cell Research | Vol 22 No 3 | March 2012