Table10_Acute changes in free and extracellular vesicle-associated circulating miRNAs and myokine profile in professional sky-runners during the Gran Sasso d’Italia vertical run.XLSX

The modification of gene expression profile, a first step in adaptation to exercise, leads to changes in the level of molecules associated with skeletal muscle activity and energy metabolism—such as myokines—as well as those involved in their transcriptional regulation, like microRNA. This study aimed to investigate the influence of strenuous exercise on circulating microRNAs and their possible association with myokine response. Pre-competition and post-competition plasma samples were collected from 14 male athletes participating in a vertical run (+1,000 m gain, 3,600 m length). Circulating total (t-miRNA) and extracellular vesicle-associated (EV-miRNA) miRNAs were extracted from the pooled plasma. Nanoparticle tracking analysis was performed to investigate pre- and post-competition EV concentration and size distribution. A panel of 179 miRNAs was assayed by qPCR and analyzed by Exiqon GenEx v6 normalized on the global mean. t-miRNA and EV-miRNAs whose level was ≥5-fold up- or down-regulated were validated for each single subject. Target prediction on MirWalk v3.0, Gene-Ontology, and pathway enrichment analysis on Panther v17.0 were performed to define the potential biological role of the identified miRNAs. A panel of 14 myokines was assayed in each sample by a multiplex immunoassay. In whole plasma, five miRNAs were upregulated and two were downregulated; in the EV fraction, five miRNAs were upregulated and three were downregulated. Nanoparticle tracking analysis revealed a similar EV size distribution in pre- and post-competition samples and a decreased concentration in post-competition samples related to pre-competition samples. Gene-Ontology and pathway enrichment analysis revealed that the identified t-miRNAs and EV-miRNAs were potentially involved in metabolism regulation in response to exercise. Correlation between fold-change of the post-competition relative to pre-competition plasma level of both t-miRNAs and EV-miRNAs and myokines further confirmed these results. This study provides an example of a systemic response to acute endurance exercise, in which circulating miRNAs play a pivotal role.

基因表达谱的重塑是运动适应的首要步骤，其可改变与骨骼肌活动及能量代谢相关的分子（如肌因子（myokines））水平，同时影响参与其转录调控的分子，例如微小RNA（microRNA）。本研究旨在探究力竭运动对循环微小RNA的影响，及其与肌因子应答的潜在关联。本研究招募14名男性运动员，采集其参与垂直竞速跑（海拔爬升1000米，全程3600米）前后的血浆样本。从混合血浆中提取循环总微小RNA（total miRNA, t-miRNA）以及与细胞外囊泡结合的微小RNA（extracellular vesicle-associated miRNA, EV-miRNA）。采用纳米颗粒追踪分析（nanoparticle tracking analysis, NTA）检测比赛前后细胞外囊泡的浓度与粒径分布。采用qPCR对179个微小RNA进行检测，并通过以全局均值标准化的Exiqon GenEx v6软件进行分析。对表达量上调或下调≥5倍的循环总微小RNA与细胞外囊泡结合微小RNA，在每个受试者样本中进行验证。采用MirWalk v3.0进行靶基因预测，通过基因本体（Gene-Ontology, GO）与Panther v17.0进行通路富集分析，以明确所鉴定微小RNA的潜在生物学功能。采用多重免疫分析法对所有样本中的14种肌因子进行检测。在全血浆样本中，共鉴定出5个上调、2个下调的微小RNA；在细胞外囊泡组分中则分别为5个上调与3个下调的微小RNA。纳米颗粒追踪分析结果显示，比赛前后样本的细胞外囊泡粒径分布无显著差异，但赛后样本的囊泡浓度较赛前有所降低。基因本体与通路富集分析结果表明，所鉴定的循环总微小RNA与细胞外囊泡结合微小RNA可能参与运动应答的代谢调控过程。循环总微小RNA与细胞外囊泡结合微小RNA的赛后相对于赛前血浆水平的倍数变化，与肌因子水平的相关性分析进一步验证了上述结论。本研究为急性耐力运动的系统性应答提供了范例，其中循环微小RNA发挥了关键调控作用。