Abstract: Background: Urinary extracellular vesicles (UEVs) are membrane bound particles that contain microRNAs (miRNAs) from cells in the urinary tract. Evaluation of UEV-associated miRNAs in canine urothelial carcinoma (UC) could provide novel biomarkers for UC and indicate pathways involved in tumorigenesis.
Objectives: Compare UEV-associated miRNAs of dogs with UC with those of healthy dogs and dogs with urinary tract infection (UTI - primary differential diagnosis for UC).
Animals: Discovery cohort: 12 UC dogs, 6 healthy dogs and 5 UTI dogs. Validation cohort: 8 UC dogs, 5 healthy dogs and 8 UTI dogs.
Methods: UEVs were isolated from 1-5mL of urine by ultrafiltration combined with size exclusion chromatography, prior to RNA extraction. Small RNAs were identified using next generation sequencing in the discovery cohort. Differential expression (DE) analysis of small RNA sequencing data identified candidate miRNAs, the expression of which was evaluated in both discovery and validation cohorts using digital droplet PCR (ddPCR).
Results: DE analysis identified 11 miRNAs that were upregulated in UEVs from UC dogs (vs. healthy), six of which (miR-143, miR-145, miR-150, miR-199, miR-451, miR-486) were selected for further evaluation. Increased expression of most candidate miRNAs (after normalisation to urine creatinine concentration) in UEVs of UC dogs (vs. UTI and healthy dogs) was confirmed by ddPCR in the discovery cohort, but these candidate miRNAs were not significantly over-expressed in the validation cohort.
Conclusions and clinical importance: Differentially expressed miRNAs associated with UEVs may provide insights into the pathogenesis of canine UC, however their utility as biomarkers requires further investigation.