MicroRNA-204-5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans

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.