A subset of microRNAs in the Dlk1‐Dio3 cluster regulates age‐associated muscle atrophy by targeting Atrogin‐1

Abstract

Background The microRNAs (miRNAs) down-regulated in aged mouse skeletal muscle were mainly clustered within the delta-like homologue 1 and the type III iodothyronine deiodinase (Dlk1-Dio3) genomic region. Although clustered miRNAs are coexpressed and regulate multiple targets in a specific signalling pathway, the function of miRNAs in the Dlk1-Dio3 cluster in muscle aging is largely unknown. We aimed to ascertain whether these miRNAs play a common role to regulate age-related muscle atrophy. Methods To examine anti-atrophic effect of miRNAs, we individually transfected 42 miRNA mimics in fully differentiated myotubes and analysed their diameters. The luciferase reporter assay using target 3′ untranslated region (UTR) and RNA pull-down assay were employed to ascertain the target predicted by the TargetScan algorithm. To investigate the therapeutic potential of the miRNAs in vivo, we generated adeno-associated virus (AAV) serotype 9 expressing green fluorescent protein (GFP) (AAV9-GFP) bearing miR-376c-3p and infected it into the tibialis anterior muscle of old mice. We performed morphometric analysis and measured ex vivo isometric force using a force transducer. Human gluteus maximus muscle tissues (ages ranging from 25 to 80 years) were used to investigate expression levels of the conserved miRNAs in the Dlk1-Dio3 cluster. Results We found that the majority of miRNAs (33 out of 42 tested) in the cluster induced anti-atrophic phenotypes in fully differentiated myotubes with increasing their diameters. Eighteen of these miRNAs, eight of which are conserved in humans, harboured predicted binding sites in the 3′ UTR of muscle atrophy gene-1 (Atrogin-1) encoding a muscle-specific E3 ligase. Direct interactions were identified between these miRNAs and the 3′ UTR of Atrogin-1, leading to repression of Atrogin-1 and thereby induction of eIF3f protein content, in both human and mouse skeletal muscle cells. Intramuscular delivery of AAV9 expressing miR-376c-3p, one of the most effective miRNAs in myotube thickening, dramatically ameliorated skeletal muscle atrophy and improved muscle function, including isometric force, twitch force, and fatigue resistance in old mice. Consistent with our findings in mice, the expression of miRNAs in the cluster was significantly down-regulated in human muscle from individuals > 50 years old.

The long noncoding RNAMeg3regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

“…Indeed, the phenotype of regenerating shMeg3-treated muscle conspicuously resembles age-related muscle defects (Beggs et al 2004;Lukjanenko et al 2019). Recently, a subset of Dlk1-Dio3 miRNAs have been shown to suppress age-related atrophy (Shin et al 2020), and these ncRNAs -including Meg3 -are markedly downregulated in aged muscle (Mikovic et al 2018). Given that aged satellite cells accumulate epigenetic abnormalities that suppress their ability to efficiently regenerate muscle (Liu et al 2013;Sousa-Victor et al, 2014), we postulate that age-related Meg3 downregulation in muscle induces a reprogramming of the epigenetic landscape that is more sensitized to pathological TGFβ and EMT signaling.…”

Functional Non-coding RNA During Embryonic Myogenesis and Postnatal Muscle Development and Disease

“…Ectopic expression of miR-23a was sufficient to protect the muscle from atrophy both in vitro and in vivo (Wada et al, 2011 ). Direct interactions were identified between miR-376c-3p and the 3′ UTR of Atrogin-1, leading to repression of Atrogin-1, and thereby induction of eIF3f protein levels in both human and mouse skeletal muscle cells (Shin et al, 2020 ). Reportedly, lncRNA Pvt1 is upregulated during muscle atrophy by blocking c-Myc phosphorylation and degradation.…”

Non-coding RNA basis of muscle atrophy

“…The 5 miRNAs (miR-377, miR-495, miR-1197, miR-379, and miR-376c) that were downregulated in aged mice were also negatively correlated with age in the gluteus maximus muscles from humans. 75 When the primary miRNA (pri-miRNA) and mature miRNA expression levels were compared among six elderly (70 ± 2 years) and six younger (29 ± 2 years) men in a study of skeletal muscle biopsies, it was found that pri-miRNA-1-1, pri-miRNA-1-2, pri-miRNA-133a-1, and pri-miRNA-133a-2 were upregulated in the elderly; however, mature miRNA-1 and miRNA-133a were not disturbed. 76 , 77 …”