miR-182, miR-221 and miR-222 are potential urinary extracellular vesicle biomarkers for canine urothelial carcinoma
Urothelial carcinoma (UC) is the most common tumour type in canine bladder cancer. Current diagnostic methods are technically challenging or can lack specificity, hence there is a need for novel biomarkers of UC. To this end, we analysed the microRNA (miRNA) cargo of extracellular vesicles (EVs) from urine samples of dogs with UC to identify miRNAs that could be utilised as biomarkers. Urine was fractionated using ultrafiltration combined with size-exclusion chromatography and small RNA sequencing analysis was performed on both the EV enriched and (EV free) protein fractions. A greater number of candidate miRNA biomarkers were detected in the EV fraction than the protein fraction, and further validation using droplet digital PCR (ddPCR) was performed on the EV enriched fraction of a second cohort of dogs with bladder cancer which validated three miRNAs as candidate biomarkers; miR-182, miR-221 and miR-222 as being significantly overrepresented in dogs with UC when compared with healthy dogs and dogs with urinary tract infections. Pathway analysis confirmed that these three miRNAs are involved in cancer. In addition, their potential downstream gene targets were predicted and PIK3R1, a well-known oncogene is likely to be a shared target between miRNA-182 and miRNA-221/222. In summary, this study highlights the potential of urinary EV-associated miRNAs as a source of biomarkers for the diagnosis of canine UC. Overall design: To find new biomarkers for canine urithelial carcinoma from extracellular vesicle and protein SEC fraction samples isolated from dog urine. Sample groups include urothelial carcinoma (UC), urinary tract infection (UTI) and healty control (HC) samples. Eleven samples were analysed as replicates and two libraries were perepared from same RNA.