Circulating microRNAs as potential predictive biomarkers of equine metabolic syndrome.

Equine Metabolic Syndrome (EMS) is a complex endocrine disorder characterized by insulin dysregulation (ID), and an increased risk of developing laminitis, often accompanied by generalized or regional adiposity. Despite growing awareness of EMS, early and accurate diagnosis remains challenging. In this pilot study, we investigated changes in the levels of circulating microRNAs (miRNAs) in plasma samples from horses diagnosed with EMS (n = 13), compared to overweight/obese (OW/OB) non-ID horses (n = 16) and healthy controls (n = 16). Using a targeted OpenArray, followed by a Kruskal-Wallis and Dunn's post hoc tests, we identified four miRNAs that were differentially expressed across the groups: miR-221, which was increased in EMS group vs healthy (p value= 0.013), miR-132, which was increased in OW/OB non-ID group vs both EMS and healthy (p value= 0.048 for both comparisons) and, miR-186 and miR-15a, which were increased in healthy controls vs EMS (p value = 0.019 for miR-186 and p value = 0.032 for miR-15a) and OW/OB non-ID (p value = 0.001 for miR-186 and p value = 0.009 for miR-15a). Functional enrichment analysis of their putative target genes revealed involvement in key insulin signalling mechanisms, including phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT), AMP-activated protein kinase (AMPK) and forkhead box O protein (FOXO) signalling pathways. Receiver Operating Characteristic (ROC) curve analysis demonstrated that combinations of these miRNAs provided discriminatory power between clinical groups (AUCs ranging from 0.79 to 0.93). These findings support the potential of circulating miRNA levels as non-invasive predictive biomarkers for EMS development. Moreover, the signalling pathways identified provide mechanistic insight and establish a foundation for future studies aimed at therapeutic target discovery.