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Simple Summary: Hypoxia drives the dysregulation of RNA molecules in canine oral melanoma (COM), influencing tumor progression and metastasis. While hypoxia-associated miRNAs have been explored, this study focused on other non-coding RNAs (ncRNAs) using NGS and qPCR. The present study revealed significant hypoxia-regulated alterations in ncRNA expression profiles, highlighting ENSCAFT00000084705.1 as consistently downregulated in COM tissues and cell lines compared to healthy tissue. Notably, its absence in plasma and extracellular vesicles suggests limited biomarker potential. This study provides evidence of transcriptional changes in ncRNAs except for miRNAs in COM, highlighting ENSCAFT00000084705.1 as a promising candidate for future investigations into the role of the transcriptome in the hypoxia-driven progression of this aggressive cancer. 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. [ABSTRACT FROM AUTHOR]

Novel small non‐coding RNAs (sRNAs) represent an emerging line of research in both human and canine oncology, due to their diverse regulatory and functional roles. Novel sRNAs are regarded as distinct from microRNAs, although both are part of the exosomal cargo. Recently, we reported on exosomal miRNAs as biomarkers for canine melanoma; however, it is unknown if novel sRNAs hold similar potential. Accordingly, we aimed to identify and validate novel sRNAs as potential biomarkers of canine oral melanoma, as part of our larger project on sequencing small exosomal RNA for this disease. Next generation sequencing revealed several differentially expressed novel sRNAs in exosomes from two melanoma cell lines (KMeC and LMeC) when compared with reference exosomes (from tumour‐free dogs). Among these novel sRNAs, long noncoding RNA fragments, tRNA‐derived fragments, snoRNAs and snRNAs were abundantly expressed. We selected four novel sRNAs upregulated in each cell line, and validated their aberrant expression with qPCR. In analysis using plasma‐derived exosomes from melanoma patients, six out of the eight selected novel sRNAs showed significantly elevated expression. Receiver operating curve (ROC) analysis showed that one long non‐coding RNA‐derived small fragment (ENSCAFT00000069599.1) and one transfer RNA‐derived small fragment (tRNA‐Ala‐TGC‐5‐1) have more than 85% sensitivity and specificity for differentiating melanoma patients from tumour‐free dogs. Therefore, we consider that novel sRNAs may serve as candidate biomarkers to facilitate more accurate diagnosis of canine oral melanoma in clinical settings. [ABSTRACT FROM AUTHOR]

Background/Aim: Canine mammary gland tumors (MGTs), as a potential model of human breast cancer, have a well-defined histological classification system. MicroRNA (miRNA) expression is a key part of the molecular signatures of both MGTs and human breast cancer, although the signatures alone do not yet provide a sufficient basis for definitive diagnosis. In this study, we investigated the association between miRNA expression patterns and histological classification. Materials and Methods: Mammary gland tissue was collected from healthy dogs (n=7) and dog patients (n=80). Further samples (n=5) were obtained from established MGT cell lines. We targeted miRNAs differentially expressed in metastatic tumor tissue versus non-metastatic and normal tissue. A subset of samples was analyzed using small RNA next generation sequencing (NGS) with subsequent qPCR. Results: Six differentially expressed miRNAs were selected from the NGS analysis and submitted for large-scale qPCR. The large-scale qPCR analysis revealed greater alternations in miRNA expression. Large-scale analysis, based on 79 samples, revealed a hierarchical clustering based on selected miRNAs that did not strikingly match the histopathological subtype classification. Conclusion: We successfully investigated the large-scale miRNA expression pattern in canine MGT and provided the whole miRNA expression. The selected miRNA demonstrated that there is no straightforward mapping between molecular signatures and histological classification of canine MGTs at the miRNA level. [ABSTRACT FROM AUTHOR]

Considering the importance of the canine cancer model of human disease, as well as the need for strategies for canine cancer management, the properties of exosomes are an emerging topic in canine oncology. In our study, exosomal RNA was isolated and investigated by next‐generation sequencing. We identified several differentially expressed microRNAs (miRNAs/miRs) in the exosomes of two melanoma cell lines compared with non‐tumor reference exosomes. We explored these potential melanoma‐specific exosomal miRNAs further and found that miR‐143 and let‐7b increased in primary, whereas miR‐210, 708, 221, and 222 increased in metastatic site originated melanoma cells. Further analysis showed miR‐143 and 221 significantly increased in plasma exosomes of metastatic melanoma patients. Moreover, the sensitivity and specificity are >85% for differentiating the non‐metastatic and metastatic patients. Therefore, these miRNAs can be an incredible biomarker candidate to identify metastatic melanoma and facilitate a better prognosis. [ABSTRACT FROM AUTHOR]

Among small non‐coding RNAs (sncRNAs/sRNAs), the functional regulation of microRNAs (miRNAs) has been studied in canine oral melanoma (COM). However, the expression level of other sncRNAs, like small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), transfer RNA‐derived fragments (tRFs) and PIWI‐interacting RNAs (piRNAs), in COM is unknown. The aim of this study was to investigate sncRNAs other than miRNAs in COM from our small RNA sequencing project (PRJNA516252). We found that several snRNAs and piRNAs were upregulated, whereas tRFs and snoRNAs were downregulated in COM. Upregulation of U1 snRNA and piR‐972, and downregulation of tRNA‐ser (1) and snoRA24 was confirmed in dog melanoma tissue and cell lines by quantitative reverse transcription PCR. Consistently, the expression of tRNA‐ser (1) and snoRA24 in plasma of COM cases was also decreased. Finally, we found a similar expression trend of U1 and snoRA24 in the human cutaneous melanoma cell line, MEWO, compared with human epidermal melanocyte cells (HEMa‐Lp). In our study, snRNA, snoRNA, tRFs and piRNA were dysregulated during melanoma progression. Moreover, the melanoma‐associated expression of U1 and snoRA24 was similar in human and dog melanoma. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) dysregulation contribute the cancer pathogenesis. However, the miRNA profile of canine oral melanoma (COM), one of the frequent malignant melanoma in dogs is still unrevealed. The aim of this study is to reveal the miRNA profile in canine oral melanoma. MiRNAs profile of oral tissues from normal healthy dogs and COM patients were compared by next-generation sequencing. Along with tumour suppressor miRNAs, we report 30 oncogenic miRNAs in COM. The expressions of miRNAs were further confirmed by quantitative real-time PCR (qPCR). Pathway analysis showed that deregulated miRNAs impact on cancer and signalling pathways. Three oncogenic miRNAs targets (miR-450b, 301a, and 223) from human study also were down-regulated in COM and had a significant negative correlation with their respective miRNA. Furthermore, we found that miR-450b expression is higher in metastatic cells and regulated MMP9 expression through a PAX9-BMP4-MMP9 axis. In silico analysis indicated that miR-126, miR-20b, and miR-106a regulated the highest numbers of differentially expressed transcription factors with respect to human melanoma. Chromosomal enrichment analysis revealed the X chromosome was enriched with oncogenic miRNAs. We comprehensively analyzed the miRNA's profile in COM which will be a useful resource for developing therapeutic interventions in both species. [ABSTRACT FROM AUTHOR]

• We identified miRNAs in canine mammary gland tumors using next generation sequencing with clinical samples. • Cfa-miR-1-3p and cfa-miR-133 family were downregulated in mammary gland tumor tissue (across histological subtypes). • GO and KEGG enrichment suggested cfa-miR-1/cfa-miR-133 family regulation of RNA polymerase II core promoter transcription. Canine mammary gland tumors are very common and represent a potential model of human breast cancer, and microRNA (miRNAs) are promising biomarkers and therapeutic targets for these tumors. Accordingly, we aimed to identify miRNAs differentially expressed in canine mammary gland tumors using next generation sequencing (NGS), with subsequent confirmatory qPCR and target gene analyses. Mammary gland tissue was collected from healthy dogs (n=7) and dogs with suspected tumors (n=80). A subset of samples was analyzed with NGS to identify differentially expressed miRNAs with CLC Genome Workbench. Normal (n=10), tumor-adjacent (n=6), and tumor-bearing (n=76) mammary gland tissue samples were analyzed for the identified miRNAs using qPCR. An in silico analysis (TargetScan) was performed to predict the miRNAs' target genes using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (DAVID). We identified four miRNAs (cfa-miR-1-3p, cfa-miR-133a-3p, cfa-miR-133b-3p, and cfa-miR-133c-3p) as down regulated in canine mammary gland tumor tissues relative to normal and tumor adjacent tissues. KEGG analysis revealed the potential target genes of cfa-miR-1-3p are related to the Rap1 signaling pathway, adherens junction, and Ras signaling pathway, and those of the miR-133 family are related to the TGF-beta signaling pathway, synaptic vesicle cycle, and sphingolipid signaling pathway. In combination, these target genes are related to the regulation of transcription and DNA binding transcription (GO analysis), and the Hippo signaling pathway, adherens junction, and endocytosis (KEGG analysis). Accordingly, we suggest these four miRNAs are promising potential biomarker candidates for canine mammary gland tumors warranting further investigation. [ABSTRACT FROM AUTHOR]

• Hypoxia regulated miRNAs (HRMs) in melanoma were identified. • Hypoxic response of HRMs are different between primary and metastatic melanoma cells. • MiR-21 and 301a lead hypoxic pathways in melanoma. MicroRNAs (miRNAs) can rapidly respond to cellular stresses, such as hypoxia. This immediate miRNA response regulates numerous genes and influences multiple signaling pathways. Therefore, identifying hypoxia-regulated miRNAs (HRMs) is important in canine oral melanoma (COM) to investigate their clinical significance. The hypoxic and normoxic miRNA profiles of two COM cell lines were investigated by next generation sequencing. HRMs were identified by comparing miRNA expression profiles in these cell lines with that in COM tissue. The HRM profile was different between cell lines of primary and metastatic origin, except for miR-301a and miR-8884. The time course of miRNA expression determined by qRT-PCR, especially for miR-210 and miR-301a, showed that metastatic cells are more resistant to hypoxia than primary cells. Analysis of an experimentally validated human miRNA target database revealed that miR-21 and miR-301a control a complex gene regulatory network in response to hypoxia, which includes pathways of well-known oncogenes, such as VEGF , PTEN , and TGFBR2. In conclusions, we revealed the HRM of COM. Moreover, our study shows the difference in regulation and response of hypoxic miRNAs between primary and metastatic originated melanoma cells. [ABSTRACT FROM AUTHOR]