Identification of altered salivary microRNAs in Cavalier King Charles Spaniels affected by mitral valve disease at different ACVIM stages

Myxomatous mitral valve disease (MMVD) in Cavalier King Charles Spaniels (CKCSs) can be diagnosed at a young age. Early and sensitive biomarkers related to the disease are currently lacking. MicroRNAs (miRNAs) are involved in the onset and progression of canine MMVD. This study aimed to profile the salivary miRNAs associated with MMVD severity in CKCSs and to identify their target genes. This was a pilot prospective, cross-sectional study. Saliva was collected from twenty-five CKCSs belonging to the American College of Veterinary Internal Medicine (ACVIM) stages A (n. 6), B1 (n. 13), and B2 (n. 6), and salivary miRNAs’ expression was profiled by Next Generation Sequencing. miRNAs’ target genes were identified using bioinformatic tools. Results showed that 25 miRNAs were differentially expressed (DE) between ACVIM A and B1 dogs; 35 DE-miRNAs were modulated in ACVIM B2 compared to B1 CKCSs; 4 miRNAs were up-regulated in ACVIM stage B1 patients compared to both ACVIM stages A and B2. These findings confirm that salivary miRNAs can be successfully quantified in CKCSs and that their expression differs across MMVD severity groups. The DE-miRNAs were associated with signalling pathways related to cellular metabolism, survival, and early inflammatory remodeling. Given the exploratory and cross-sectional design, these miRNAs should be considered candidate, non-invasive indicators associated with disease severity rather than established biomarkers. Further longitudinal and validation studies are needed to assess their diagnostic potential.