BACKGROUND Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex-miRNAs (e.g. miR-1, miR-133a, miR-206, and miR-483) are highly up-regulated in the serum of DMD patients and dystrophic animal models and are restored to wild-type levels following exon skipping-mediated dystrophin rescue in mdx mice. As such, ex-miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex-miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle. METHODS Candidate ex-miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide-PMO (PPMO) exon skipping conjugates and in mdx-XistΔhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-XistΔhs mice using the FirePlex methodology and key results validated by small RNA TaqMan RT-qPCR. The muscles from each animal model were further characterized by dystrophin western blot and immunofluorescence staining. RESULTS The restoration of ex-myomiR abundance observed following PPMO treatment was not recapitulated in the high dystrophin-expressing mdx-XistΔhs group, despite these animals expressing similar amounts of total dystrophin protein (~37% of wild-type levels). Instead, ex-miRNAs were present at high levels in mdx-XistΔhs mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-XistΔhs muscles showed non-homogeneous dystrophin staining and sporadic regenerating foci. CONCLUSIONS Uniform dystrophin expression is required to prevent ex-miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle.

中文翻译：

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需要均匀的肌膜肌营养不良蛋白表达来防止细胞外微小RNA的释放并改善营养不良的病理学。

背景技术杜氏肌营养不良症（DMD）是一种由肌营养不良蛋白的遗传损失引起的致命性的肌肉萎缩性疾病。细胞外microRNA（ex-miRNA）是DMD的公认微创生物标志物。特定的ex-miRNA（例如miR-1，miR-133a，miR-206和miR-483）在DMD患者和营养不良的动物模型的血清中高度上调，并在外显子跳跃后恢复为野生型水平。介导的肌营养不良蛋白在mdx小鼠中的拯救。因此，前miRNA是有希望的外显子跳跃功效的药效生物标志物。在这里，我们旨在确定ex-miRNA水平反映营养不良性肌中肌营养不良蛋白表达的基本水平的程度。方法研究了通过肽-PMO（PPMO）外显子跳跃缀合物恢复了肌营养不良的mdx小鼠和表达X-染色体失活导致出生时可变数量的肌营养不良蛋白的mdx-XistΔhs小鼠的mdx小鼠的候选ex-miRNA生物标记水平。使用FirePlex方法对mdx-XistΔhs小鼠进行miRNA分析，并通过小RNA TaqMan RT-qPCR验证了关键结果。通过抗肌萎缩蛋白western印迹和免疫荧光染色进一步表征每种动物模型的肌肉。结果在表达高肌营养不良蛋白的mdx-XistΔhs组中，PPMO处理后观察到的肌原纤维丰富度的恢复并未得到概括，尽管这些动物表达的肌营养不良蛋白总含量相似（约占野生型水平的37％）。反而，无论抗肌萎缩蛋白的表达如何，ex-miRNA均以高水平存在于mdx-XistΔhs小鼠中。PPMO处理的肌肉表现出肌营养不良蛋白定位的均匀模式，并且没有再生纤维，而mdx-XistΔhs肌肉表现出肌营养不良蛋白染色不均一且散在的再生灶。结论需要均匀的肌营养不良蛋白表达来防止ex-miRNA释放，稳定肌纤维更新并减轻营养不良性肌肉的病理。