Table4_Identification of Potential circRNA-microRNA-mRNA Regulatory Network in Skeletal Muscle.XLSX

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.

环状RNA（circRNAs）是一类通过回文剪接新近发现的调控性RNA家族。通过对circRNA的基因组范围分析发现，circRNA在生物体内广泛表达，通过充当RNA结合蛋白（RBPs）和微小RNA（miRNAs）的“海绵”，调控基因表达。为鉴定小鼠骨骼肌中表达的circRNAs，我们对富含circRNA的腓肠肌RNA样本进行了高通量RNA测序，鉴定出超过1200种circRNAs。此外，在衰老的人类骨骼肌组织和卫星细胞中，我们分别鉴定出超过14,000和15,000种circRNAs。通过对丰富circRNA亚组进行逆转录PCR、Sanger测序和RNase R消化实验，验证了其在小鼠骨骼肌组织中的表达。circRNA-miRNA-mRNA调控网络的解析揭示了保守的circNfix可能与miR-204-5p结合，后者是肌细胞增强因子2c（Mef2c）表达的抑制因子。为了支持circNfix可能通过调控Mef2c表达来调节肌生成的假设，适度沉默circNfix降低了Mef2c mRNA的表达并抑制了C2C12细胞的分化。我们提出，circNfix在部分通过充当miR-204-5p的“海绵”作用来促进肌肉细胞分化过程中MEF2C的表达。