Table_5_The miRNome of canine invasive urothelial carcinoma.XLSX

Urothelial carcinoma (UC) comprises up to 2% of all naturally occurring neoplasia in dogs and can be challenging to diagnose. MicroRNAs (miRNAs) have been reported to be dysregulated in numerous diseases, including neoplasia. MiRNA expression has been evaluated in human UC, but there is limited information regarding the miRNA transcriptome of UC in dogs. Our study aimed to evaluate differential miRNA expression in bladder tissue collected from normal canine urothelium and canine invasive UC (iUC) to elucidate the dysregulated pathways in canine UC. Next-Generation RNA sequencing (RNA-Seq) was performed for dogs with UC (n = 29) and normal canine urothelium (n = 4). Raw RNA data were subjected to normalization, and pairwise comparison was performed using EdgeR with Benjamini-Hochberg FDR multiple testing correction (p < 0.05; >2-fold change) comparing tissue samples of normal urothelium to canine iUC samples. Principal component analysis and hierarchical cluster analysis were performed. MiRNA of FFPE tissue samples of separate iUC (n = 5) and normal urothelium (n = 5) were used to evaluate five miRNAs using RT-qPCR. Pathway analysis was performed utilizing miRWalk, STRING database, and Metascape utilizing KEGG pathways and GO terms databases. Twenty-eight miRNAs were differentially expressed (DE) by RNA-Seq. RT-qPCR confirmed that four miRNAs are significantly downregulated in UC compared to healthy urothelial samples (miR-105a, miR-143, miR-181a, and miR-214). Principal component analysis and hierarchical cluster analysis showed separation between miRNAs in iUC and the control group. The DE miRNAs are most often associated with gene silencing by miRNA, miRNAs in cancer, and miRNAs involved in DNA damage responses. Proteins involved include HRAS, KRAS, ARAF, RAF1, MAPK1, MAP2K1, MAPK3, FGFR3, EGFR, HBEGF, RASSF1, E2F2, E2F3, ERBB2, SRC, MMP1, and UP3KA. The differential expression of miRNAs in canine iUC compared to normal canine urothelial tissue indicates that these markers should be further evaluated for their potential role as diagnostic and therapeutic targets.

尿路上皮癌（Urothelial carcinoma, UC）在犬类所有自发性肿瘤中占比可达2%，临床诊疗颇具挑战。微RNA（MicroRNAs, miRNAs）已被证实可在包括肿瘤在内的多种疾病中出现表达失调。目前针对人类尿路上皮癌的miRNA表达已开展相关研究，但关于犬尿路上皮癌的miRNA转录组的报道仍较为匮乏。本研究旨在对正常犬尿路上皮与犬侵袭性尿路上皮癌（invasive urothelial carcinoma, iUC）患者的膀胱组织进行miRNA差异表达分析，以阐明犬尿路上皮癌的失调通路。本研究针对29例犬尿路上皮癌样本与4例正常犬尿路上皮样本开展了下一代RNA测序（RNA-Seq）。对原始RNA测序数据进行标准化处理后，采用EdgeR软件结合贝叶斯-霍赫贝格错误发现率（FDR）多重检验校正方法（p < 0.05，差异倍数>2），对正常尿路上皮组织与犬iUC组织样本进行两两比较分析。随后开展了主成分分析与层次聚类分析。另外，选取5例独立的犬iUC福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded, FFPE）组织样本与5例正常尿路上皮FFPE样本，通过实时定量聚合酶链反应（RT-qPCR）验证5种miRNA的表达水平。借助miRWalk、STRING数据库以及Metascape平台，结合京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路与基因本体（Gene Ontology, GO）术语数据库开展通路富集分析。RNA-Seq结果显示，共鉴定出28种差异表达miRNAs（DE miRNAs）。RT-qPCR验证结果表明，相较于正常尿路上皮样本，犬尿路上皮癌组织中有4种miRNA显著下调，分别为miR-105a、miR-143、miR-181a以及miR-214。主成分分析与层次聚类分析结果显示，iUC组与对照组的miRNA表达谱存在明显分离。上述差异表达miRNAs主要参与miRNA介导的基因沉默、癌症相关miRNA调控以及DNA损伤应答等生物学过程，其调控的靶蛋白主要包括HRAS、KRAS、ARAF、RAF1、MAPK1、MAP2K1、MAPK3、FGFR3、EGFR、HBEGF、RASSF1、E2F2、E2F3、ERBB2、SRC、MMP1以及UP3KA。犬侵袭性尿路上皮癌与正常犬尿路上皮组织间的miRNA差异表达特征提示，上述miRNA分子有望作为犬尿路上皮癌的诊断与治疗靶点，有待进一步开展相关研究验证。