Abstract
Circular RNAs (circRNAs) are a newly discovered family of regulatory RNAs generated through backsplicing. Genome-wide profiling of circRNAs found that circRNAs are ubiquitously expressed and regulate gene expression by acting as a sponge for RNA-binding proteins (RBPs) and microRNAs (miRNAs). To identify circRNAs expressed in mouse skeletal muscle, we performed high-throughput RNA-sequencing of circRNA-enriched gastrocnemius muscle RNA samples, which identified more than 1,200 circRNAs. In addition, we have identified more than 14,000 and 15,000 circRNAs in aging human skeletal muscle tissue and satellite cells, respectively. A subset of abundant circRNAs was analyzed by RT-PCR, Sanger sequencing, and RNase R digestion assays to validate their expression in mouse skeletal muscle tissues. Analysis of the circRNA-miRNA-mRNA regulatory network revealed that conserved circNfix might associate with miR-204-5p, a suppressor of myocyte enhancer factor 2c (Mef2c) expression. To support the hypothesis that circNfix might regulate myogenesis by controlling Mef2c expression, silencing circNfix moderately reduced Mef2c mRNA expression and inhibited C2C12 differentiation. We propose that circNfix promotes MEF2C expression during muscle cell differentiation in part by acting as a sponge for miR-204-5p.
Highlights
Skeletal muscle is the largest organ contributing to one-third of the human body weight
The growth media of the sub-confluent C2C12 and Human skeletal muscle myoblasts (HSMM) cells were replaced with the differentiation media (DMEM supplemented with 2% horse serum), and the cells were differentiated into myotubes for up to 4 days
The total number of circRNAs identified in aged skeletal muscle was significantly higher than in young samples (Figure 1B)
Summary
Skeletal muscle is the largest organ contributing to one-third of the human body weight. In addition to the well-known myogenic transcription factors, noncoding RNAs such as microRNAs, lncRNAs, and the recently discovered circular RNAs (circRNAs) were involved in muscle cell differentiation and muscle development (Zhao et al, 2019; Das et al, 2020). We identified circRNAs expressed in young and aged skeletal muscles. We predicted their association with muscle miRNAs and downstream target genes. We analyzed the previously published RNA-sequencing data from young and old human skeletal muscle and satellite cells to identify age-associated circRNAs. Together, our study provides new information to better understand the age-associated decline in muscle function and develop a new therapeutic strategy for ageassociated muscle diseases
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