Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle. Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle

Exercise affects the expression of microRNAs (miR/s) and muscle-derived extracellular vesicles (EVs). To evaluate sarcoplasmic and secreted miR expression in human skeletal muscle in response to exercise-mimetic contractile activity, we utilized a three-dimensional tissue-engineered model of human skeletal muscle (“myobundles”). Myobundles were subjected to three culture conditions: no electrical stimulation (CTL), chronic low frequency stimulation (CLFS), or intermittent high frequency stimulation (IHFS) for 7 days. RNA was isolated from myobundles and from extracellular vesicles (EVs) secreted by myobundles into culture media; miR abundance was analyzed by miRNA-sequencing. We used edgeR and a within-sample design to evaluate differential miR expression and Pearson correlation to evaluate correlations between myobundle and EV populations within treatments with statistical significance set at p<0.05. Numerous miRs were differentially expressed between myobundles and EVs; 116 miRs were differentially expressed within CTL, 3 within CLFS, and 2 within IHFS. Additionally, 25 miRs were significantly correlated (18 in CTL, 5 in CLFS, 2 in IHFS) between myobundles and EVs. Electrical stimulation resulted in differential expression of 8 miRs in myobundles and only 1 miR in EVs. Several KEGG pathways, known to play a role in regulation of skeletal muscle, were enriched, with differentially overrepresented miRs between myobundle and EV populations identified using miEAA. Together, these results demonstrate that in vitro exercise-mimetic contractile activity of human engineered muscle affects both their expression of miRs and number of secreted EVs. These results also identify novel miRs of interest for future studies of the role of exercise in organ-organ interactions in vivo Overall design: Differential microRNA expression analysis of miRNA-seq data for human tissue-engineered muscle and extracellular vesicles these tissues secrete

运动可调控微小核糖核酸（microRNAs, miR/s）与肌肉衍生细胞外囊泡（muscle-derived extracellular vesicles, EVs）的表达水平。为探究人类骨骼肌在模拟运动的收缩刺激下，其肌浆与分泌型miR的表达变化，本研究采用人类骨骼肌三维组织工程模型——"肌束（myobundles）"开展实验。实验将肌束分为三种培养处理组：无电刺激对照组（no electrical stimulation, CTL）、慢性低频刺激组（chronic low frequency stimulation, CLFS）、间歇性高频刺激组（intermittent high frequency stimulation, IHFS），各组刺激时长均为7天。分别从肌束以及肌束分泌至培养基的细胞外囊泡中提取总RNA，通过miRNA测序（miRNA-sequencing）分析miR的表达丰度。本研究采用edgeR软件与样本内设计分析差异miR表达情况，通过Pearson相关分析评估各处理组内肌束与EV群体间的表达相关性，统计学显著性阈值设定为p<0.05。结果显示，肌束与EV间存在大量差异表达的miR：其中CTL组共鉴定出116个差异表达miR，CLFS组3个，IHFS组2个。此外，肌束与EV间共有25个miR呈显著相关关系，其中CTL组18个、CLFS组5个、IHFS组2个。电刺激处理可使肌束中8个miR发生差异表达，而EV中仅1个miR出现差异表达。通过miEAA工具鉴定得到肌束与EV群体间差异高表达的miR，其富集的多条KEGG通路已被证实参与骨骼肌的生理调控过程。综上，本研究结果证实：人类工程化肌肉在体外（in vitro）模拟运动的收缩刺激下，其miR表达水平与分泌的EV数量均会发生改变。本研究同时鉴定出若干新型miR，可为未来探索运动在体内（in vivo）器官间互作中的调控作用提供潜在研究靶点。整体实验设计：针对人类组织工程化肌肉及其分泌的细胞外囊泡的miRNA测序数据，开展差异微小核糖核酸表达分析。