Figure 2.

miR-21 induces cardiocyte outgrowth and down-regulation of SPRY2. (a) The stem-loop structure of miR-21 under the control of the CMV promoter cloned into recombinant Ad, in which the mature sequence is shown in red. (b) The anti-miR-21 (miR-21 eraser) sequence under the control of the U6 promoter cloned into recombinant adenovirus. The panels below each construct represent Northern blots of RNA extracted from cardiocytes infected with 10–20 moi of the corresponding virus for 48 h (n = 3). (c) Cardiocytes cultured on uncoated glass chamber slides were infected with 10–20 moi of adenovirus expressing miR-21 (n = 20) or short hairpin SPRY2 (shRNA-SPRY2) in serum-free medium (n = 6). (d) Similarly cells were treated with 10–20 moi of control or a combination of miR-21 and SPRY2 Ad viruses (n = 4). Seventy-two hours later, cells were fixed and coimmunolabeled with anti-SPRY2 (green), anti-titin (red), and DAPI (blue). Cells were imaged with an epifluorescence microscope at 60× magnification by using an fluorescein isothiocyanate (FITC) (green), Texas Red (red), or FITC/Texas Red/DAPI triple filter, as shown. NC, noncardiac cell. (e–h) Cardiocytes were infected with Ad expressing miR-21 (n = 4; e), shRNA-SPRY2 (n = 4; f), miR-21 eraser (n = 4; g), or SPRY2 (n = 4; h) and control Ad expressing scrambled miR, or lacz, respectively, for 48 h, in serum-free medium, as marked with the + sign. Cells were additionally treated with 10% FBS for 10 min, where indicated. Control lanes (unmarked with +) were treated with a scrambled stem-loop– (e–g) or a Lacz (h)—expressing virus. Cells were harvested, and the cytosolic fraction was analyzed by Western blotting using anti-SPRY2, anti-phosoho-p42/p44 (p-p42/p44), anti-p42/p44, anti-GAPDH, and anti-α sarcomeric actin (s.actin). (i) Cardiocytes were transfected with a CMV.Luc vector containing the predicted miR-21 target sequence in SPRY2 (Luc-SPRY2) or a control mutated sequence (Luc-mtSPRY2), within the 3′UTR of Luc, where indicated by the + sign. In conjunction, cells were cotransfected with plasmids expressing miR-21 (red bars) or a control nonsense stem-loop sequence (open bars). After 24 h, protein was extracted, and luciferase activity per microgram of protein was measured and averaged (n = 12). The results are graphed as -fold increase in luciferase activity relative to Luc-SPRY2 in the absence of miR-21 after adjusting it to 1. Error bars represent SE of the mean, and *p < 0.001, Luc-SPRY2 + miR-21 versus Luc-SPRY2 alone; **p < 0.0001, Luc-SPRY2 versus Luc-mtSPRY2.