Differential expression of miRNAs in experimental metacarpal and lumbar wounds in horses.

Differential expression of microRNAs (miRNAs) in wounds from distinct anatomical regions (metacarpal vs. lumbar) of horses suggests region-specific regulatory mechanisms impacting wound healing and possibly the development of exuberant granulation tissue in distal limb wounds.

Introduction and Background

• Delayed healing and exuberant granulation tissue (EGT) commonly occur in distal limb wounds of horses.
• EGT formation is linked to hypoxia (low oxygen) and persistent inflammation in the wound area.
• MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression post-transcriptionally and are involved in many biological processes, including wound healing.
• However, the specific involvement and profiles of miRNAs during equine wound healing, especially across different body sites, had not been well characterized before this study.

Study Objective

• To investigate whether miRNA expression differs between wounds located on the metacarpal (distal limb) compared to the lumbar (back) region in horses.
• Hypothesis: Different anatomical sites have distinct miRNA expression profiles influencing the healing response.

Methodology

• Subjects: Four healthy adult horses.
• Creation of standardized full-thickness skin wounds (2 × 2 cm) at two locations per horse: metacarpal (distal limb) and lumbar region (back).
• Sample collection: Tissue biopsies taken 21 days after wounding, representing an intermediate-to-late phase of healing.
• Sample processing: RNA isolated from formalin-fixed paraffin-embedded (FFPE) samples, which preserves tissue morphology and nucleic acids.
• miRNA profiling performed using Affymetrix miRNA 4.1 microarray platform to assess the expression of many miRNAs simultaneously.
• Statistical analysis used empirical moderation of variance and considered miRNAs with p-values < 0.05 and fold change ≥ +2 as significantly differentially expressed.
• Sequence comparison to human miRNAs via miRBase was conducted to identify orthologs and infer potential functional roles.

Results

• Identified six miRNAs that showed differential expression between metacarpal and lumbar wounds.
• eca-miR-205 was significantly upregulated in the metacarpal wounds compared to lumbar wounds.
• The remaining five miRNAs—eca-miR-127, eca-miR-379, eca-miR-335, eca-miR-376c, and eca-miR-487b—were all downregulated in the metacarpal wounds relative to lumbar wounds.
• These differences suggest location-specific post-transcriptional regulatory mechanisms mediated by miRNAs that may affect wound healing outcomes.

Interpretation and Significance

• This is the first study to identify specific miRNAs differentially expressed during wound healing in horses, demonstrating anatomical site-dependent regulation.
• Higher eca-miR-205 in distal limb wounds aligns with known roles of miR-205 in epithelial repair and inflammation modulation in other species.
• Reduced expression of the other five miRNAs in metacarpal wounds may contribute to the delayed healing and increased granulation tissue characteristic of distal limb wounds by impacting pathways related to inflammation, cell proliferation, and extracellular matrix remodeling.
• Understanding miRNA profiles provides insight into why distal limb wounds heal poorly compared to wounds in the lumbar region.
• These miRNAs hold potential as biomarkers to predict wound healing prognosis or targets for therapeutic intervention to improve healing and reduce pathological tissue formation.

Conclusions

• miRNAs play distinct roles depending on wound location in horses, impacting healing outcomes.
• Region-specific miRNA expression profiles underscore the importance of site-tailored approaches in managing equine wounds.
• Further research could explore mechanistic pathways influenced by these miRNAs and test their utility as biomarkers or therapeutic targets to improve healing of challenging distal limb wounds in horses.