Differential microRNA Expression during Muscle Regeneration. Mus musculus

MicroRNAs (miRNAs) are important in the regulation of many biological processes including muscle development. However, little is known regarding miRNA regulation of muscle regeneration. In mature murine tibialis anterior muscle following injury, 298 miRNAs were significantly changed during the time course of muscle regeneration including 86 that were altered greater than 10-fold as compared to uninjured muscle. Temporal miRNA expression patterns were identified and included inflammation-related miRNAs (miR-223 and -147) that increased immediately after injury; this pattern contrasted to that of mature muscle-specific miRNAs (miR-1, -133a and -499) that were abruptly decreased following injury and then up-regulated in later regenerative events. Another cluster of miRNAs were transiently increased in the early days of muscle regeneration. This included miR-351, a miRNA that was also transiently expressed during myogenic progenitor cell (MPC) differentiation in vitro. Based on computational predictions, further studies demonstrated that E2f3 was a target of miR-351 in myoblasts. Moreover, knockdown of miR-351 expression inhibited MPC proliferation and promoted apoptosis during MPC differentiation, whereas miR-351 overexpression protected MPC from apoptosis during differentiation. Collectively, these observations suggest that miR-351 is involved in both the maintenance of MPC proliferation and the transition of MPC into differentiated myotubes. Thus, a novel, time-dependent sequence of molecular events during skeletal muscle regeneration has been identified, i.e., miR-351 inhibits E2f3 expression, a key regulator of cell cycle progression and proliferation, and promotes MPC proliferation and protects early differentiating MPC from apoptosis, important events in the hostile tissue environment after acute muscle injury. Overall design: Skeletal muscles are damaged and repaired repeatedly throughout life. Muscle regeneration maintains locomotor function during aging and delays the appearance of clinical symptoms in neuromuscular diseases, such as Duchenne muscular dystrophy. The capacity for skeletal muscle growth and regeneration is conferred by satellite cells located between the basal lamina and the sarcolemma of mature myofibers. Upon injury, satellite cells reenter the cell cycle, proliferate, and then exit the cell cycle either to renew the quiescent satellite cell pool or to differentiate into mature myofibers. Despite recent advances, genes involved in these processes are still largely unknown. Understanding the molecular mechanisms that regulate satellite cell activities could promote development of novel countermeasures to enhance muscle regeneration that is compromised by diseases or aging. Using a muscle injury mouse model, we profiled miRNA expression during muscle regeneration.

微小RNA（microRNAs，miRNAs）在包括肌肉发育在内的诸多生物学过程的调控中发挥关键作用。然而，目前针对miRNA调控肌肉再生的分子机制尚缺乏深入认知。在损伤后的成熟小鼠胫前肌中，肌肉再生时序过程内共有298种miRNA发生显著表达变化，其中86种的表达幅度较未损伤肌肉超过10倍。
研究鉴定出三类具有时序特征的miRNA表达模式：其一为炎症相关miRNA（miR-223与miR-147），在损伤后即刻表达上调；该模式与成熟肌肉特异性miRNA（miR-1、miR-133a及miR-499）的表达模式截然相反，后者在损伤后迅速下调，随后在再生后期事件中表达上调。另有一类miRNA在肌肉再生早期呈瞬时上调表达，其中包括miR-351——该miRNA在体外肌源性祖细胞（myogenic progenitor cell，MPC）分化过程中同样呈瞬时表达。
基于生物信息学预测，后续研究证实E2f3是成肌细胞中miR-351的靶基因。此外，敲低miR-351的表达会抑制MPC增殖，并在MPC分化过程中促进细胞凋亡；而过表达miR-351则可在分化阶段保护MPC免受凋亡。综上，上述结果表明miR-351既参与维持MPC的增殖状态，也介导MPC向分化成熟肌管的转化过程。由此，本研究鉴定出骨骼肌再生过程中一类全新的时序依赖性分子事件序列：miR-351通过抑制细胞周期进程与增殖的关键调控因子E2f3的表达，促进MPC增殖，并保护早期分化的MPC免受凋亡——这在急性肌肉损伤后的恶劣组织微环境中是至关重要的事件。
研究设计：
骨骼肌在生命周期中会反复经历损伤与修复过程。肌肉再生可在衰老过程中维持运动功能，并延缓杜氏肌营养不良症（Duchenne muscular dystrophy）等神经肌肉疾病的临床症状发作。骨骼肌生长与再生的能力由位于成熟肌纤维基膜（basal lamina）与肌膜（sarcolemma）之间的卫星细胞（satellite cell）所赋予。当肌肉受到损伤时，卫星细胞会重新进入细胞周期并增殖，随后退出细胞周期，要么自我更新以维持静息卫星细胞库，要么分化为成熟肌纤维。尽管近年来相关研究取得了一定进展，但参与上述过程的基因仍大多未被阐明。解析调控卫星细胞活性的分子机制，有望助力开发全新的干预策略，以增强因疾病或衰老而受损的肌肉再生能力。本研究通过肌肉损伤小鼠模型，对肌肉再生过程中的miRNA表达谱进行了系统性分析。