Robriquet Florence, Babarit Candice, Larcher Thibaut, Dubreil Laurence, Ledevin Mireille, Goubin Hélicia, Rouger Karl, Guével Laëtitia
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.
BMC Musculoskelet Disord. 2016 May 11;17:209. doi: 10.1186/s12891-016-1060-5.
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.
杜氏肌营养不良症（DMD）是一种X连锁肌肉疾病，可导致纤维坏死和进行性麻痹。目前，DMD仍然是一种致命疾病，尚无任何有效治疗方法，需要更好地了解其病理生理过程并对新发现的治疗策略进行全面评估。包括肌肉特异性肌微RNA家族成员在内的微RNA已被确定在DMD患者的肌肉以及用作DMD模型的mdx小鼠肌肉中表达失调。近年来，金毛寻回犬型肌营养不良症（GRMD）犬已成为客观评估新创新方法潜力的关键动物模型。在此，我们首先旨在确定这一临床相关模型中5种选定微RNA的肌肉表达模式，以确定与其他DMD情况相比，它们是否受到类似影响。其次，我们试图证明这些微RNA是否会受到一种有前景的干细胞候选物（称为MuStem细胞）全身递送的影响，以深化我们对其作用模式的认识和/或识别与细胞治疗疗效相关的标志物。
使用逆转录定量聚合酶链反应（RT-qPCR）和原位杂交技术，对9月龄健康犬以及GRMD犬的三个亚组（未进行免疫抑制或细胞移植、接受持续免疫抑制方案、在免疫抑制下接受MuStem细胞移植）的微RNA表达水平和细胞定位进行了比较研究。
我们发现，与健康犬相比，GRMD犬肌肉中miR-222的表达明显上调，而miR-486的表达则趋于下调。有趣的是，miR-1、miR-133a和miR-206的表达没有变化。原位杂交研究首次揭示，miR-486和miR-206主要定位于GRMD犬肌肉中新再生的纤维中。此外，我们表明，同种异体细胞移植中常用的基于环孢素的免疫抑制仅影响miR-206的表达。最后，我们证明动脉内注射MuStem细胞会导致miR-133a和miR-222上调，同时与miR-222靶标的两种肌节蛋白表达下调。
我们指出miR-222和miR-486在肌肉中的表达差异与临床相关GRMD犬模型的病理生理学相关，其组织定位集中在再生纤维上。我们还确定了MuStem细胞输注后miR-133a和miR-222的表达变化。
