Varvil Mara S, Bailey Taylor, Dhawan Deepika, Knapp Deborah W, Ramos-Vara José A, Dos Santos Andrea P
Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, United States.
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, United States.
Front Vet Sci. 2022 Aug 22;9:945638. doi: 10.3389/fvets.2022.945638. eCollection 2022.
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 ( = 29) and normal canine urothelium ( = 4). Raw RNA data were subjected to normalization, and pairwise comparison was performed using EdgeR with Benjamini-Hochberg FDR multiple testing correction ( < 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 ( = 5) and normal urothelium ( = 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（UC）占犬类自然发生的所有肿瘤的2%，诊断具有挑战性。据报道，微小RNA（miRNA）在包括肿瘤在内的多种疾病中表达失调。miRNA表达已在人类UC中进行评估，但关于犬类UC的miRNA转录组的信息有限。我们的研究旨在评估从正常犬尿路上皮和犬浸润性UC（iUC）收集的膀胱组织中miRNA的差异表达，以阐明犬类UC中失调的途径。对患有UC的犬（n = 29）和正常犬尿路上皮（n = 4）进行了下一代RNA测序（RNA-Seq）。对原始RNA数据进行标准化处理，并使用EdgeR和Benjamini-Hochberg FDR多重检验校正（P < 0.05；变化倍数>2倍）进行成对比较，比较正常尿路上皮组织样本与犬iUC样本。进行了主成分分析和层次聚类分析。使用RT-qPCR对单独的iUC（n = 5）和正常尿路上皮（n = 5）的FFPE组织样本中的miRNA进行评估，以评估5种miRNA。利用miRWalk、STRING数据库和Metascape，利用KEGG通路和GO术语数据库进行通路分析。通过RNA-Seq有28种miRNA差异表达（DE）。RT-qPCR证实，与健康尿路上皮样本相比，4种miRNA在UC中显著下调（miR-105a、miR-143、miR-181a和miR-214）。主成分分析和层次聚类分析显示iUC中的miRNA与对照组之间存在分离。差异表达的miRNA最常与miRNA介导的基因沉默、癌症中的miRNA以及参与DNA损伤反应的miRNA相关。涉及的蛋白质包括HRAS、KRAS、ARAF、RAF1、MAPK1、MAP2K1、MAPK3、FGFR3、EGFR、HBEGF、RASSF1、E2F2、E2F3、ERBB2、SRC、MMP1和UP3KA。与正常犬尿路上皮组织相比，犬iUC中miRNA的差异表达表明，这些标志物作为诊断和治疗靶点的潜在作用应进一步评估。
