Figure 2. miR-222 regulates cardiomyocyte hypertrophy, proliferation and apoptosis in vitro.

By using miR-222 gain- and loss-of-function in NRVMs, miR-222’s effects on cardiac cell size, proliferation, and hypertrophic markers were examined. A and B: Immunohistochemical staining for sarcomeric α-actinin followed by quantification of cardiomyocyte area as described in methods. Cells were transfected with control or miR-222 precursor in A and with control antimiR (ctl-anti) or antimiR-222 (anti-222) in B. At least 200 cells were quantified in each group. These data demonstrate miR-222 is necessary and sufficient to induce cardiomyocyte hypertrophy. C. Quantification of EdU and Ki67 staining, as well as cell number from primary NRVMs transfected with control precursor (ctl-pre) or miR-222 precursor (pre-222). D. Quantification of EdU and Ki67 staining, and cell number from NRVMs transfected with control antimiR (ctl-anti) or antimiR-222 (anti-222). These data demonstrate miR-222 is necessary and sufficient to induce proliferation of NRVMs. E. Flow cytometry analysis of TUNEL staining in cardiomyocytes treated with control precursor (ctl-pre) or miR-222 precursor (pre-222) or control antimiR (ctl-anti) or antimiR-222 (anti-222). Cardiomyocye apoptosis was induced by serum deprivation (SD) or serum deprivation plus doxorubicin (SD+doxorubicin). These data demonstrate miR-222 inhibits cardiomyocyte apoptosis. F. QRT-PCR for markers of cardiomyocyte hypertrophy and/or pathology in NRVMs treated with control (ctl-pre) or miR-222 precursor (pre-222). These data demonstrate that miR-222 induces a physiological pattern of gene expression. Data are shown as mean±SEM fold-change of gene expression normalized to U6 and reflect at least three independent experiments. Scale bar: 100 μm. *p<0.05, **p<0.01 versus respective control using Student’s test.