Abstract 17262: MicroRNA-23b Mediated Inhibition of Gsk-3β and Nischarin Promotes Proliferation of Cardiomyocytes
Background: Promoting proliferation of cardiomyocytes (CMs) following myocardial infarction is a promising approach for cardiac regeneration. Recent studies from our lab identified a panel of microRNAs inducing proliferation of adult CMs; of these miR-23b was one of the most robust CM proliferation-inducing miR and is conserved across species. In silico analysis identified binding sites for miR-23b on the 3’UTRs of both GSK-3β (GSK) and nischarin (NISCH). Here, we hypothesize that miR-23b promotes both in vitro and in vivo proliferation of CMs by regulating the expression of cell cycle inhibitors GSK and NISCH.
Methods and Results: Adult CMs isolated from 8-12 week old female rats were transfected with hsa-miR-23b, cel-miR-67 (control), siGSK, siNISCH, siGSK + siNISCH (si=siRNA). EdU was added (5 μM) each day post-transfection and cells were fixed on day 6 for IF staining (Troponin-I, EdU, pH3, and DAPI), protein, and RNA analysis. A significant increase in EdU+ CMs was found in cells transfected with miR-23b (8.51%±0.78), siGSK (8.30%±1.31) and siNISCH (5.73%±1.52) when compared to control miR cel-67 (3.13%±0.84). Interestingly, simultaneous inhibition of both GSK and NISCH exhibited robust proliferation (13.88%±1.15) similar to miR-23b transfection. Luciferase assay confirmed binding sites for miR-23b on the 3’UTR of GSK with a 0.85±0.04 fold reduction of chemiluminescent activity. Compared to control, western blotting following miR-23b transfection revealed an increase in pERK (2.46±0.69 fold), a decrease in GSK (0.18±0.12 fold), and an increase in β-catenin (4.17±2.03 fold), indicating miR-23b mediated inhibition of GSK and subsequent increase in β-catenin. For in vivo analysis, 6 day old Fischer rats received intraperitoneal adenoviral miR-23b (10^11 viral particles) followed by EdU injections on alternate days. On day 6, rats were sacrificed, and the hearts were fixed/sectioned for IF staining. Rats injected with miR-23b expressed higher levels of EdU (2.42±0.46 fold), and decreased fold of GSK (0.32±0.10) and NISCH (0.29±0.17) compared to control as confirmed via IHC staining.
Conclusion: MiR-23b promotes proliferation of CMs by simultaneous inhibition of both GSK and NISCH and upregulation of downstream targets pERK and β-catenin.
Author Disclosures: J.S. Crocker: None. R. Pandey: None. R.P. Ahmed: None.
- © 2015 by American Heart Association, Inc.