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. 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.

尿路上皮癌（Urothelial carcinoma, UC）是犬膀胱癌最常见的肿瘤类型。当前临床诊断方法要么技术门槛较高，要么特异性不足，因此亟需开发针对尿路上皮癌的新型生物标志物。为此，我们对患尿路上皮癌的犬只尿液样本中的细胞外囊泡（extracellular vesicles, EVs）所载的微小RNA（microRNA, miRNA）进行分析，以筛选可作为诊断标志物的miRNA。我们采用超滤联合尺寸排阻色谱法对尿液进行分级分离，并分别对EV富集组分及无EV蛋白质组分开展小RNA测序分析。相较于蛋白质组分，EV组分中检测到的候选miRNA生物标志物数量更多。随后，我们针对第二组患膀胱癌的犬只的EV富集组分，采用液滴数字PCR（droplet digital PCR, ddPCR）开展验证实验，确认miR-182、miR-221及miR-222可作为候选生物标志物：与健康犬及患尿路感染（urinary tract infection, UTI）的犬只相比，患尿路上皮癌的犬只体内这三种miRNA的表达水平显著上调。通路分析证实，这三种miRNA均参与癌症发生相关的生物学过程。此外，我们预测了这三种miRNA的潜在下游靶基因，发现已知致癌基因PIK3R1很可能是miR-182与miR-221/222的共同靶基因。综上，本研究证实了尿液EV相关miRNA作为犬尿路上皮癌诊断生物标志物来源的潜力。本数据集旨在从犬尿液分离得到的细胞外囊泡及蛋白质尺寸排阻色谱（size-exclusion chromatography, SEC）分级组分中，筛选犬尿路上皮癌的新型生物标志物。样本分组包括尿路上皮癌（UC）组、尿路感染（UTI）组及健康对照（healthy control, HC）组。本研究对11份样本开展了重复分析，并从同一份RNA样本中构建了两个测序文库。