Comprehensive Analysis of miRNAs and Predicted Protein Interaction Networks in Skeletal Muscle Development of Myostatin‐Deficient Rabbits

ABSTRACT Myostatin (MSTN), encoded by the MSTN gene, is a critical negative regulator of skeletal muscle mass. This study aims to identify and characterize the miRNAs involved in the development of the double‐muscling phenotype in MSTN ‐deficient rabbits. We performed high‐throughput sequencing to analyze the miRNA expression profiles in gluteus maximus tissue from wild type ( MSTN +/+ ) and MSTN ‐KO ( MSTN +/− and MSTN −/− inclusive) rabbits. Differentially expressed miRNAs (DEmiRNAs) were identified, and their potential target genes were predicted. Functional enrichment analysis of these target mRNAs was conducted using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to elucidate the involved biological pathways and regulatory networks. A total of 25 DEmiRNAs (13 downregulated and 12 upregulated, |log 2 FC| ≥ 1.0, adjusted p < 0.05) and 1178 differentially expressed mRNAs (408 upregulated and 770 downregulated, |log 2 FC| ≥ 2.0, adjusted p < 0.05) were identified in MSTN ‐KO compared to MSTN +/+ rabbits. Bioinformatics analysis revealed that the target genes of these DEmiRNAs were significantly enriched in key pathways governing muscle growth and metabolism, including the PI3K‐Akt signaling pathway, MAPK signaling pathway, and pathways related to ECM‐receptor interaction and insulin signaling. Notably, many predicted target mRNAs are expressed by genes that encode key inhibitors of myogenesis (e.g., HDAC4 ) and major extracellular matrix components (e.g., COL4A3, POSTN ). Our results demonstrate that MSTN deficiency induces a distinct and widespread change in the miRNA expression landscape of skeletal muscle.