Angiogenesis-related microRNAs and signalling pathways in canine mammary tumours

Angiogenesis, a hallmark of cancer, supports tumour growth and metastasis by establishing an abnormal vascular network, and microRNAs (miRNAs) regulate this process post-transcriptionally. Because evidence in canine mammary tumours (CMTs) remains limited, we profiled 24 putative pro- and anti-angiogenic miRNAs by RT-qPCR in benign and malignant CMTs compared with normal mammary glands, and we predicted angiogenesis-related targets using multiMiR followed by Gene Ontology and KEGG pathway enrichment analyses. Intratumoral angiogenesis was quantified as microvascular density (MVD) and endothelial area (EA) on Factor VIII-immunolabeled sections using QuPath. MVD and EA were higher in malignant than in benign CMTs and peaked in grade III carcinomas. Malignant CMTs showed a progressive shift towards a pro-angiogenic miRNA profile, with significant upregulation of pro-angiogenic miR-9, miR-20a, miR-98, miR-210, and miR-21(p < 0.05). Conversely, anti-angiogenic miRNA displayed a heterogenous, context-dependent expression pattern: miR-152-3p and miR-542-3p were downregulated in benign CMTs relative to normal mammary tissue, whereas miR-205 and miR-34a were upregulated in malignant CMTs (p < 0.05). In malignant CMTs, MVD correlated with EA (r = 0.8, p = 0.0003), EA correlated with miR-98 (r = 0.67, p = 0.006), and tumour size correlated with miR-210 (r = 0.58, p = 0.03). In benign tumours, EA correlated with miR-497 (r = 0.81, p = 0.02). Target prediction identified 16,910 genes, with pro- and anti-angiogenic miRNAs sharing 86.5% of predicted targets, indicating extensive regulatory overlap. KEGG enrichment highlighted 100 significantly enriched pathways (FDR < 0.05), including MAPK, PI3K-Akt, HIF-1, VEGF, and breast cancer signalling, with MAPK1 and MAPK3 among the most frequently targeted genes. Finally, miR-34a showed the best diagnostic performance for distinguishing benign from malignant CMTs. Overall, findings support a substantial contribution of miRNAs to angiogenic regulation in CMTs, strengthen the utility of the canine model in comparative breast cancer research, and highlight the potential of miRNA-based biomarkers for tumour stratification and anti-angiogenic targeting.