Various microRNAs (miRNAs) present in autologous blood products of canines have not been studied recently. We aimed to elucidate the existence of miRNAs in platelet-rich fibrin (PRF) and the stability of canine autologous blood products under various storage conditions. Total RNAs were isolated from PRF and other autologous blood products following newly adapted protocols used in commercial kits for plasma and tissue samples. Quantitative real-time polymerase chain reaction analysis (qPCR) was used to detect miRNAs in autologous blood products. The miR-16, miR-21, miR-155, and miR-146a were abundant in PRF and other autologous blood products of canines. Furthermore, we found they could maintain stability under protracted freezing temperatures of −30 °C for at least one month. Our findings revealed that PRF might be a stable resource for various canine miRNAs.

Hypoxia contributes to tumor progression and metastasis, and hypoxically dysregulated RNA molecules may, thus, be implicated in poor outcomes. Canine oral melanoma (COM) has a particularly poor prognosis, and some hypoxia-mediated miRNAs are known to exist in this cancer; however, equivalent data on other hypoxically dysregulated non-coding RNAs (ncRNAs) are lacking. Accordingly, we aimed to elucidate non-miRNA ncRNAs that may be mediated by hypoxia, targeting primary-site and metastatic COM cell lines and clinical COM tissue samples in next-generation sequencing (NGS), with subsequent qPCR validation and quantification in COM primary and metastatic cells and plasma and extracellular vesicles (EVs) for any identified ncRNA of interest. The findings suggest that a number of non-miRNA ncRNA species are hypoxically up- or downregulated in COM. We identified one ncRNA, the long ncRNA fragment ENSCAFT00000084705.1, as a molecule of interest due to its consistent downregulation in COM tissues, hypoxically and normoxically cultured primary and metastatic cell lines, when compared to the oral tissues from healthy dogs. However, this molecule was undetectable in plasma and plasma EVs, suggesting that its expression may be tumor tissue-specific, and it has little potential as a biomarker. Here, we provide evidence of hypoxic transcriptional dysregulation for ncRNAs other than miRNA in COM for the first time and suggest that ncRNA ENSCAFT00000084705.1 is a molecule of interest for future research on the role of the transcriptome in the hypoxia-mediated progression of this aggressive cancer.

Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) can be difficult to differentiate but must be diagnosed correctly as treatment and prognosis for these tumors differ markedly. Relevant diagnostic biomarkers are thus needed, and those identified in dogs may have utility in human medicine because of the similarities between human and canine HCA and HCC. A tRNA-derived fragment (tRF), tRNA-Val, is a promising potential biomarker for canine mammary gland tumors but has not previously been investigated in hepatic tumors. Accordingly, we aimed to elucidate the potential utility of tRNA-Val as a biomarker for canine HCA and HCC using clinical samples (tumor tissue and plasma extracellular vesicles [EVs]) and tumor cell lines with qRT-PCR assays. We also investigated relevant functions and signaling pathways with bioinformatic analyses (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes). tRNA-Val was markedly downregulated in HCC tumor tissue versus HCA tumor tissue and normal liver tissue, and a similar trend was shown in plasma EVs and HCC cell lines versus healthy controls. Based on areas under the receiver operating characteristic curves (AUCs), tRNA-Val significantly distinguished HCC (AUC = 1.00, p = 0.001) from healthy controls in plasma EVs and HCC from HCA (AUC = 0.950, p = 0.01). Bioinformatics analysis revealed that tRNA-Val may be primarily involved in DNA repair, mRNA processing, and splicing and may be linked to the N-glycan and ubiquitin-mediated proteasome pathways. This is the first report on the expression of tRNA-Val in canine HCC and HCA and its possible functions and signaling pathways. We suggest that tRNA-Val could be a promising novel biomarker to distinguish canine HCC from HCA. This study provides evidence for a greater understanding of the role played by tRNA-Val in the development of canine HCC.

Canine hepatocellular carcinoma (HCC) requires further molecular characterization to identify diagnostic and therapeutic targets, and to establish whether dogs with this condition can model the human disease. Accordingly, we aimed to identify differentially expressed genes (DEGs) in canine HCC and evaluate cross‐species transcriptomic dysregulation in canine and human HCC. Liver tissue samples from three dogs with HCC and three healthy dogs were subjected to next‐generation sequencing, followed by RT‐qPCR validation. Identified DEGs were then targeted in bioinformatics analyses (pathway enrichment, protein‐protein interaction network, and hub gene analyses) for molecular characterization and comparison with human HCC datasets. We identified 975 DEGs (upregulated: 604; and downregulated: 371). Extracellular matrix‐receptor interaction, focal adhesion, cell adhesion molecule, PI3K/Akt signaling, and cytokine/chemokine‐related pathways were enriched. C1R, APOC3, C1QA, APOA1, C1QB, ACTG1, C1QC, CRP, ANXA5, and ANXA2 were identified as hub genes. Canine and human HCCs share 118 DEGs, highlighting conserved alterations in metabolic pathways, PI3K‐Akt signaling, focal adhesion, and PPAR signaling pathways. Based on human HCC data, SPP1, NQO1, RRM2, APOA1, APOC3, ALDOB, and IGF1 were identified as prognosticators indicating poor overall survival. This study presents the first cross‐species transcriptomic analysis of canine HCC, revealing significant molecular resemblances to human HCC, indicating it may be a promising comparative model for studying tumor biology, drug responses, and novel therapeutic interventions.