Using an unbiased high‐throughput microRNA (miRNA)‐silencing screen combined with functional readouts for mitochondrial oxidative capacity in C2C12 myocytes, we previously identified 19 miRNAs as putative regulators of skeletal muscle mitochondrial metabolism. In the current study, we highlight miRNA‐204‐5p, identified from this screen, and further studied its role in the regulation of skeletal muscle mitochondrial function. Following silencing of miRNA‐204‐5p in C2C12 myotubes, gene and protein expression were assessed using quantitative polymerase chain reaction, microarray analysis, and western blot analysis, while morphological changes were studied by confocal microscopy. In addition, miRNA‐204‐5p expression was quantified in human skeletal muscle biopsies and associated with in vivo mitochondrial oxidative capacity. Transcript levels of PGC‐1α (3.71‐fold; p  < .01), predicted as an miR‐204‐5p target, as well as mitochondrial DNA copy number (p < .05) and citrate synthase activity (p = .06) were increased upon miRNA‐204‐5p silencing in C2C12 myotubes. Silencing of miRNA‐204‐5p further resulted in morphological changes, induced gene expression of autophagy marker light chain 3 protein b (LC3B; q = .05), and reduced expression of the mitophagy marker FUNDC1 (q  = .01). Confocal imaging revealed colocalization between the autophagosome marker LC3B and the mitochondrial marker OxPhos upon miRNA‐204‐5p silencing. Finally, miRNA‐204‐5p was differentially expressed in human subjects displaying large variation in oxidative capacity and its expression levels associated with in vivo measures of skeletal muscle mitochondrial function. In summary, silencing of miRNA‐204‐5p in C2C12 myotubes stimulated mitochondrial biogenesis, impacted on cellular morphology, and altered expression of markers related to autophagy and mitophagy. The association between miRNA‐204‐5p and in vivo mitochondrial function in human skeletal muscle further identifies miRNA‐204‐5p as an interesting modulator of skeletal muscle mitochondrial metabolism.

中文翻译：

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MicroRNA-204-5p调节C2C12肌管中的线粒体生物发生，并与人类的氧化能力相关。

通过使用无偏高通量microRNA（miRNA）沉默筛选结合功能读数来检测C2C12心肌细胞中线粒体的氧化能力，我们先前确定了19种miRNA作为骨骼肌线粒体代谢的假定调节剂。在当前的研究中，我们突出显示了从该筛选中鉴定出的miRNA-204-5p，并进一步研究了其在骨骼肌线粒体功能调节中的作用。在C2C12肌管中沉默miRNA-204-5p后，使用定量聚合酶链反应，微阵列分析和蛋白质印迹分析评估基因和蛋白质表达，同时通过共聚焦显微镜研究形态变化。此外，在人类骨骼肌活检中定量了miRNA‐204‐5p的表达，并与体内线粒体的氧化能力有关。p   <.01）被预测为miR‐204‐5p的靶标，并且线粒体DNA拷贝数（p  <.05）和柠檬酸合酶活性（p = .06）在C2C12中通过miRNA‐204‐5p沉默后增加肌管。miRNA-204-5p沉默进一步导致形态学改变，诱导自噬标记轻链3蛋白b（LC3B; q = .05）的基因表达以及线粒体标记FUNDC1（q  = .01）。共聚焦成像显示，miRNA-204-5p沉默后，自噬标记物LC3B与线粒体标记物OxPhos共定位。最后，miRNA-204-5p在人类受试者中差异表达，显示出氧化能力及其体内表达水平与骨骼肌线粒体功能相关的大变化。总之，在C2C12肌管中沉默miRNA-204-5p刺激线粒体生物发生，影响细胞形态，并改变与自噬和线粒体相关的标志物的表达。miRNA-204-5p与人体骨骼肌的体内线粒体功能之间的关联进一步确定了miRNA-204-5p是骨骼肌线粒体代谢的有趣调节剂。