MicroRNA-486 overexpression delays the disease pathology of muscular dystrophy. M. S. Alexander1,2, J. C. Casar3, N. Motohashi4, N. M. Vieira1,2, I. Eisenberg5, J. L. Marshall1,2, M. J. Gasperini1, A. Lek1,2, J. A. Myers1, E. A. Estrella1,6, P. B. Kang1,6,7, F. Shapiro8, F. Rahimov1,2, G. Kawahara1,2, J. J. Widrick1, L. M. Kunkel1,2,9,10 1) Division of Genetics and Genomics at Boston Childrens Hospital, Boston, MA 02115; 2) Department of Pediatrics and Genetics at Harvard Medical School, Boston, MA 02115; 3) Departamento de Neurología, Escuela de Medicina, Pontificia Universidad Católica de Chile. Santiago, RM, Chile; 4) Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School, Minneapolis, MN 55455; 5) Center for Human Placenta Research, Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center- Mt. Scopus Jerusalem, Israel; 6) Department of Neurology, Boston Childrens Hospital and Harvard Medical School, Boston, MA 02115; 7) Present Address: Division of Pediatric Neurology, University of Florida College of Medicine, Gainsville, FL 32610; 8) Departments of Orthopedic Surgery at Boston Childrens Hospital and Harvard Medical School, Boston MA 02115; 9) The Manton Center for Orphan Disease Research at Boston Childrens Hospital, Boston, MA 02115; 10) Harvard Stem Cell Institute, Cambridge, MA 02138.

   Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in the dysregulation of many signaling pathways that interact directly or indirectly with the dystrophin protein. Previously, we identified miR-486 as being strongly reduced in its expression levels in the dystrophin-deficient mouse and muscle biopsies of human DMD patients. Here we report that transgenic overexpression of the muscle-enriched microRNA, miR-486, in mdx5cv (dystrophin-mutant) mice resulted in improved serum biochemistry, reduced apoptosis, increased myofiber size, and improved muscle physiological force output. Using a bioinformatic approach, we identified DOCK3, dedicator-of-cytokinesis-3, as being a direct downstream target of miR-486 in skeletal muscle. Manipulation of DOCK3 expression in myoblast cell culture had strong effects on normal and DMD myoblast apoptosis, and on the RAC1/RHOA signaling pathway. Overexpression of DOCK3 resulted in myoblast apoptosis and reduced myoblast fusion, and activated RAC1/RHOA signaling in normal muscle cells. Conversely, while overexpression of DOCK3 in DMD muscle cells induced myoblast apoptosis and reduced myoblast fusion, the RAC1/RHOA signaling pathway was not activated. Overexpression of miR-486 in DMD myoblasts resulted in a restoration of normal RAC1/RHOA signaling most likely due increased muscle membrane stability and reduced muscle apoptosis. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates many of the signs of the disease pathology of dystrophin-deficient muscle.

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