Knockdown of circFOXN3 Promotes the Proliferation and Differentiation of Bovine Myoblasts

Circular RNAs (circRNAs) are emerging as pivotal regulators of skeletal muscle development, a process critical to meat production in livestock. However, the specific functions of most circRNAs in bovine myogenesis remain unexplored. This study investigates the role of a novel circRNA, circFOXN3, in the proliferation and differentiation of bovine myoblasts. We first confirmed the circular nature of circFOXN3 by identifying its specific back-splice junction via divergent primers and Sanger sequencing, and its resistance to RNase R digestion as well as stability under actinomycin D treatment were confirmed. Subcellular fractionation assays revealed that circFOXN3 is predominantly localized in the cytoplasm. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that circFOXN3 was highly and specifically expressed in muscle tissues and exhibits dynamic expression patterns during myoblast proliferation and differentiation. Functional knockdown of circFOXN3 using specific small interfering RNAs (siRNAs) during the proliferation phase significantly enhanced myoblast proliferation, as evidenced by elevated expression of proliferation marker genes (PCNA, CCND2) and increased 5-ethynyl-2'-deoxyuridine (EdU) incorporation. RNA-seq analysis following circFOXN3 knockdown revealed a comprehensive set of differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis reveal the significant enrichment in critical pathways, most notably the cell cycle pathway. Furthermore, protein-protein interaction (PPI) analysis of DEGs identified several core nodes in the cell cycle pathway, including CDC20, CDK1, BUB1, and CCNA2. Conversely, knockdown during the differentiation phase promoted myogenic differentiation, confirmed by the upregulation of differentiation markers (MyoG, MyoD1) and enhanced myotube formation. Our findings provide the first functional evidence that circFOXN3 acts as a potent negative regulator of bovine myogenesis. Its silencing promotes myoblast expansion and terminal differentiation, likely by modulating key cell cycle pathways and gene expression networks via its cytoplasmic localization. These results position circFOXN3 as a promising molecular target for genetic strategies aimed at improving muscle growth and efficiency in cattle production.