Expression analysis in multiple muscle groups and serum reveals complexity in the microRNA transcriptome of the mdx mouse and implications for therapy

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions including Duchenne muscular dystrophy (DMD). We have compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx mouse relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as a number of changes common between tissues, including previously identified dystromirs. In the case of miR-31 and miR-34c, up-regulation of primary-miRNA transcripts, precursor hairpins and all mature miRNAs derived from the same transcript or miRNA cluster strongly suggests transcriptional regulation of these miRNAs. The most striking differences in differential miRNA expression were between muscle tissue and serum. Specifically, miR-1, miR-133a and miR-206 were highly abundant in serum but down-regulated or modestly up-regulated in muscle, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalised in response to peptide-PMO mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable in the development of novel therapeutics and for monitoring their efficacy. Male WT C57/B10 and male mdx C57/B10 mice (n=4) were sacrificed at eight weeks of age. The heart, quadriceps femoris, and diaphragm were dissected and flash frozen in liquid nitrogen-cooled isopentane. Samples were homogenised using Precellys 24 (Bertin Technologies, France), and RNA was extracted using TRIzol reagent (Invitrogen) as according to the manufacturer's instructions.微RNA（miRNAs）是一类调控基因表达的小型RNA分子，广泛参与多种细胞进程与病理状态，包括杜氏肌营养不良症（Duchenne muscular dystrophy, DMD）。本研究对比了mdx小鼠与野生型对照小鼠的肢体近端肌肉、肢体远端肌肉、膈肌、心脏及血清中的差异miRNA表达情况。全局转录组分析揭示了差异miRNA表达的肌肉特异性模式，同时还发现了多个组织间共有的表达变化，包括此前已被鉴定的营养不良相关miRNA（dystromirs）。针对miR-31与miR-34c，其初级miRNA转录本、前体发夹结构以及同一转录本或miRNA簇产生的所有成熟miRNA均出现上调，这强烈提示这些miRNA受到转录水平调控。差异miRNA表达最显著的差异存在于肌肉组织与血清之间：具体而言，miR-1、miR-133a及miR-206在血清中含量极高，但在肌肉中则呈下调或轻度上调状态，这表明这些miRNA是极具潜力的疾病生物标志物。事实上，经肽-PMO介导的肌营养不良蛋白恢复疗法治疗后，这些miRNA在血清中的相对水平恢复至正常范围。本研究揭示了mdx小鼠miRNA转录组的更为复杂的表达特征，对其的深入理解将有助于新型治疗策略的开发以及治疗效果的监测。本研究选取8周龄的雄性野生型C57/B10小鼠与雄性mdx C57/B10小鼠各4只（n=4），处死后对心脏、股四头肌及膈肌进行解剖分离，并置于液氮冷却的异戊烷中快速冷冻；随后使用Precellys 24研磨仪（法国 Bertin Technologies公司）对样本进行匀浆处理，并按照制造商说明书采用TRIzol试剂（Invitrogen公司）提取总RNA。