Upregulation and functional roles of miR-450b in canine oral melanoma.

Canine oral melanoma (COM) is a common and highly aggressive disease with the potential to model human melanomas. Dysregulated microRNAs represent an interesting line of research for COM because they are implicated in tumor progression. One example is miR-450b, which has been investigated for its molecular mechanisms and biological functions in multiple human cancers, but not human or canine melanoma. Here, we aimed to investigate miR-450b as a potential diagnostic biomarker of COM and its functional roles in metastatic and non-metastatic forms of the disease. We investigated the expression of miR-450b and its target mRNA genes in clinical (tumor tissue and plasma) samples and metastatic and primary-tumor cell lines. Knockdown and overexpression experiments were performed to determine the influence of miR-450b on cell proliferation, migration, colony formation, and apoptosis. miR-450b was significantly upregulated in COM and differentiated between metastatic and non-metastatic tumors, and its potential as a biomarker of metastatic and non-metastatic COM was further confirmed in ROC analysis. miR-450b knockdown promoted cell proliferation, migration, and clonogenicity and inhibited apoptosis, whereas its overexpression yielded the reverse pattern. miR-450b directly binds 3' UTR of PAX9 mRNA and modulates its function leading to BMP4 downregulation and MMP9 upregulation at the transcript level. Furthermore, we surmised that miR-450b activates the Wnt signaling pathway based on gene ontology and enrichment analyses. We concluded that miR-450b has the potential as a diagnostic biomarker and could be a target candidate for COM treatment.

Hypoxia-related Y RNA fragments as a novel potential biomarker for distinguishing metastatic oral melanoma from non-metastatic oral melanoma in dogs.

Hypoxia may promote tumor progression, and hypoxically altered noncoding RNA (ncRNA) expression may play a role in metastasis. Canine oral melanoma (COM) frequently metastasizes, and ncRNA expression under hypoxia may be clinically significant. We aimed to elucidate ncRNA fragments whose expression is altered by hypoxia in COM-derived primary KMeC and metastatic LMeC cell lines using next-generation sequencing to validate these results in qRT-PCR, and then compare expression between metastatic and non-metastatic COM. The NGS analysis and subsequent qRT-PCR validation were performed using hypoxic and normoxic KMeC and LMeC cells, and clinical samples [tumor tissue, plasma, and plasma-derived extracellular vesicles] obtained from dogs with metastatic or non-metastatic melanoma were analyzed with qRT-PCR. Y RNA was significantly decreased in metastatic LMeC cells versus primary KMeC cells in hypoxic and normoxic conditions. The expression of Y RNA was decreased in dogs with metastatic melanoma versus those with non-metastatic melanoma for all clinical sample types, reflecting the pattern found with hypoxia. Receiver operating characteristic analysis demonstrated that Y RNA level is a promising biomarker for discriminating metastatic from non-metastatic melanoma in plasma [area under the curve (AUC) = 0.993, p < 0.0001] and plasma-derived extracellular vesicles (AUC = 0.981, p = 0.0002). Overall, Y RNA may be more resistant to hypoxic stress in the metastatic than the non-metastatic state for COM. However, further investigation is required to elucidate the biological functions of Y RNA under hypoxic conditions.

Development of a Microminipig Model of Atherosclerosis for the Evaluation of a HMGCR Inhibitor.

BACKGROUND/AIM: Atherosclerosis is known as a major risk factor for cardiovascular disease, and development of an animal model of atherosclerosis is required to investigate its clinical pathogenesis. We studied the optimal amount of cholesterol in the diet and the optimal experimental period for development of a Microminipig model of atherosclerosis for the evaluation of a hydroxymethylglutaryl-CoA reductase (HMGCR) inhibitor (atorvastatin). MATERIALS AND METHODS: Eighteen male animals (3-4 months old) were divided into 3 groups. Group 1 consisted of control animals receiving a normal chow diet, Group 2 animals received a high fat (12% w/w) and low cholesterol (0.1% w/w) diet (HFLCD), and Group 3 animals received HFLCD+statin for 12 weeks. Animals received statin at 3 mg/kg body weight per day. HFLCD did not down-regulate the hepatic expression of HMGCR mRNA. RESULTS: HFLCD increased body, omentum, and mesenteric adipose tissue weight, and induced hypercholesterolemia and atherosclerotic lesions in the abdominal aorta. HFLCD+statin inhibited hypercholesterolemia and atherosclerotic lesions, but not obesity. CONCLUSION: A microminipig atherosclerosis model induced by HFLCD can be used in the evaluation of HMGCR inhibitors for the treatment of hypercholesterolemia and atherosclerosis.

Elevated expression of miR-301a and its functional roles in canine oral melanoma.

miR-301a is one of numerous dysregulated microRNAs (miRNAs) in canine oral melanoma (COM), one of which is miR-301a (upregulated). Its biological role has been described in various human cancer types, including malignant melanoma, but not in COM. Accordingly, in this study, we investigated miR-301a expression in COM in greater detail to ascertain whether it could serve as a diagnostic biomarker, elucidate its functional roles in this cancer, and predict the possible pathways by which it exerts its effects. Relative expression of miR-301a was investigated in clinical oral tissue and plasma samples and COM cell (KMeC and LMeC) lines using qRT-PCR. Knockdown of miR-301a was also validated for KMeC and LMeC cells using qRT-PCR. We performed CCK-8 assays to assess cell proliferation, monolayer wound-healing, and transwell migration assays to assess cell migration, a colony-formation assay to assess clonogenicity, a TUNEL assay and flow cytometry to assess apoptosis-related effects, and gene enrichment analyses to predict possible related pathways. miR-301a was markedly upregulated in COM oral tissue and plasma clinically, suggesting its potential as a diagnostic biomarker for COM diagnosis. In vitro assays demonstrated that miR-301 significantly inhibited apoptosis in COM cells while promoting cell migration, proliferation, and clonogenicity. We also predicted that miR-301 exerts cancer-promoting effects through the Wnt signalling pathway for COM. Our findings suggest that miR-301a is a COM oncomiR that regulates several oncogenic phenotypes with the potential to be a diagnostic biomarker.