title: Identification in GRMD dog muscle of critical miRNAs involved in pathophysiology and effects associated with MuStem cell transplantation.
abstract: Duchenne muscular dystrophy (DMD) is an X-linked muscle disease that leads to fibre necrosis and progressive paralysis. At present, DMD remains a lethal disease without any effective treatment, requiring a better understanding of the pathophysiological processes and comprehensive assessment of the newly identified therapeutic strategies. MicroRNAs including members of the muscle-specific myomiR family have been identified as being deregulated in muscle of DMD patients and in mdx mice used as a model for DMD. In recent years, the Golden Retriever muscular dystrophy (GRMD) dog has appeared as the crucial animal model for objectively assessing the potential of new innovative approaches. Here, we first aim at establishing the muscle expression pattern of five selected miRNAs in this clinically relevant model to determine if they are similarly affected compared with other DMD contexts. Second, we attempt to show whether these miRNAs could be impacted by the systemic delivery of a promising stem cell candidate (referred to as MuStem cells) to implement our knowledge on its mode of action and/or identify markers associated with cell therapy efficacy.
  
  
  
  A comparative study of miRNAs expression levels and cellular localization was performed on 9-month-old healthy dogs, as well as on three sub-sets of GRMD dog (without immunosuppression or cell transplantation, with continuous immunosuppressive regimen and with MuStem cell transplantation under immunosuppression), using RT-qPCR and in situ hybridization.
  
  
  
  We find that miR-222 expression is markedly up-regulated in GRMD dog muscle compared to healthy dog, while miR-486 tends to be down-expressed. Intriguingly, the expression of miR-1, miR-133a and miR-206 does not change. In situ hybridization exploration reveals, for the first time, that miR-486 and miR-206 are mainly localized in newly regenerated fibres in GRMD dog muscle. In addition, we show that cyclosporine-based immunosuppression, classically used in allogeneic cell transplantation, exclusively impacts the miR-206 expression. Finally, we demonstrate that intra-arterial administration of MuStem cells results in up-regulation of miR-133a and miR-222 concomitantly with a down-expression of two sarcomeric proteins corresponding to miR-222 targets.
  
  
  
  We point out a differential muscle expression of miR-222 and miR-486 associated with the pathophysiology of the clinically relevant GRMD dog model with a tissue localization focused on regenerated fibres. We also establish a modified expression of miR-133a and miR-222 subsequent to MuStem cell infusion.
authors.affiliation: INRA UMR 703 PAnTher "Physiopathologie Animale et bioThérapie du muscle et du système nerveux", Oniris, Atlanpôle - La Chantrerie, Route du Gachet C.S. 40706, F-44307, Nantes Cedex 03, France.|LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique, F-44307, Nantes, France.|Université de Nantes, F-44322, Nantes, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Robriquet
authors.affiliation: INRA UMR 703 PAnTher "Physiopathologie Animale et bioThérapie du muscle et du système nerveux", Oniris, Atlanpôle - La Chantrerie, Route du Gachet C.S. 40706, F-44307, Nantes Cedex 03, France.|LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique, F-44307, Nantes, France.
authors.forename: Candice
authors.initials: C
authors.lastname: Babarit
authors.affiliation: INRA UMR 703 PAnTher "Physiopathologie Animale et bioThérapie du muscle et du système nerveux", Oniris, Atlanpôle - La Chantrerie, Route du Gachet C.S. 40706, F-44307, Nantes Cedex 03, France.|LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique, F-44307, Nantes, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Larcher
authors.affiliation: INRA UMR 703 PAnTher "Physiopathologie Animale et bioThérapie du muscle et du système nerveux", Oniris, Atlanpôle - La Chantrerie, Route du Gachet C.S. 40706, F-44307, Nantes Cedex 03, France.|LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique, F-44307, Nantes, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Dubreil
authors.affiliation: INRA UMR 703 PAnTher "Physiopathologie Animale et bioThérapie du muscle et du système nerveux", Oniris, Atlanpôle - La Chantrerie, Route du Gachet C.S. 40706, F-44307, Nantes Cedex 03, France.|LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique, F-44307, Nantes, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Ledevin
authors.affiliation: INRA UMR 703 PAnTher "Physiopathologie Animale et bioThérapie du muscle et du système nerveux", Oniris, Atlanpôle - La Chantrerie, Route du Gachet C.S. 40706, F-44307, Nantes Cedex 03, France.|LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique, F-44307, Nantes, France.
authors.forename: Hélicia
authors.initials: H
authors.lastname: Goubin
authors.affiliation: INRA UMR 703 PAnTher "Physiopathologie Animale et bioThérapie du muscle et du système nerveux", Oniris, Atlanpôle - La Chantrerie, Route du Gachet C.S. 40706, F-44307, Nantes Cedex 03, France.|LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique, F-44307, Nantes, France.
authors.forename: Karl
authors.initials: K
authors.lastname: Rouger
authors.affiliation: INRA UMR 703 PAnTher "Physiopathologie Animale et bioThérapie du muscle et du système nerveux", Oniris, Atlanpôle - La Chantrerie, Route du Gachet C.S. 40706, F-44307, Nantes Cedex 03, France. laetitia.guevel@univ-nantes.fr.|LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique, F-44307, Nantes, France. laetitia.guevel@univ-nantes.fr.|Université de Nantes, F-44322, Nantes, France. laetitia.guevel@univ-nantes.fr.
authors.forename: Laëtitia
authors.initials: L
authors.lastname: Guével
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology / Q000627:therapeutic use; D004195:Disease Models, Animal; D004285:Dogs; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D006801:Humans; D007165:Immunosuppression Therapy / Q000379:methods; D007166:Immunosuppressive Agents / Q000494:pharmacology / Q000627:therapeutic use; D017403:In Situ Hybridization; D007269:Injections, Intra-Arterial; D051379:Mice; D018101:Mice, Inbred mdx; D035683:MicroRNAs / Q000187:drug effects / Q000378:metabolism*; D032342:Muscle Cells / Q000378:metabolism / Q000637:transplantation*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D009137:Muscular Dystrophy, Animal / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D018995:Myosin Heavy Chains / Q000378:metabolism; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D015415:Biomarkers; D007166:Immunosuppressive Agents; D035683:MicroRNAs; D016572:Cyclosporine; D018995:Myosin Heavy Chains
keywords: Duchenne muscular dystrophy; GRMD dog; In situ hybridization; Skeletal muscle; Stem cell therapy; miRNA