MicroRNA–mRNA Networks in Skeletal Muscle of Tailored Pig Models for Dystrophinopathies

ABSTRACT Background Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X‐linked dystrophinopathies caused by mutations in the dystrophin ( DMD ) gene. A common DMD‐causing mutation in humans is exon 52 deletion ( DMD Δ52), which disrupts the reading frame and abolishes dystrophin expression. Therapeutic skipping of exon 51 or 53 can restore the reading frame, producing a truncated but functional protein and generating a BMD‐like phenotype. Porcine models recapitulating DMD Δ52 (DMD) and DMD Δ51–52 (BMD‐like) were used to identify molecular differences and condition‐specific miRNA–mRNA networks. Methods Skeletal muscle (triceps brachii) from four DMD, four BMD, and five wild‐type (WT) pigs at 3.5 months of age underwent stranded total RNA‐seq and small RNA‐seq. Differentially expressed mRNAs (|log2FC| ≥ 1, adj. p  ≤ 0.05) and miRNAs (adj. p  ≤ 0.05) were identified with DESeq2. miRNA–mRNA networks were constructed using RNAhybrid predictions (MFE  Results Compared with WT, DMD muscle exhibited 1440 upregulated and 487 downregulated genes, characterized by strong repression of structural, contractile, calcium‐handling and metabolic genes (e.g., MYBPC2 , MYL3 , MYLK2 , CACNA2D3 , CACNA2D4 ) and marked upregulation of inflammatory mediators and innate immune receptors (e.g., IL6 , IL18 , IL1R1 , CCR1/2/5 , TLR1/2/4/7/9 ). In contrast, BMD muscle showed partial restoration of these pathways and clustered closer to WT in global expression profiles. Distinct miRNA signatures were observed between DMD and BMD. Differential expression analysis identified 22 upregulated and 12 downregulated miRNAs in DMD versus WT and 36 upregulated and 21 downregulated miRNAs in BMD versus WT. Integration of miRNA and mRNA data yielded extensive regulatory networks (1013 unique pairs for upregulated miRNAs in DMD; 2679 pairs for downregulated miRNAs in BMD). Two condition‐specific miRNAs emerged as strong biomarker candidates: ssc‐miR‐296‐3p (upregulated exclusively in DMD, targeting 228 genes enriched in muscle structure and fatty acid metabolism) and ssc‐miR‐423‐5p (elevated specifically in BMD, targeting 67 genes involved in calcium signalling and tissue development). Several dysregulated miRNAs, including miR‐199a‐5p and miR‐199b, overlapped with those reported in human DMD and other muscular dystrophies. Conclusions Exon 51 skipping in the DMD Δ52 background partially restores key transcriptional programmes in skeletal muscle but does not fully normalize them to WT patterns. The identification of condition‐specific miRNAs highlights post‐transcriptional regulatory differences between DMD and BMD, positioning them as promising biomarkers and therapeutic targets. These findings underscore the translational value of porcine dystrophinopathy models for mechanistic studies and preclinical evaluation of RNA‐targeted interventions.