MicroRNA differential expression analysis in canine visceral hemangiosarcoma formalin-fixed, paraffin-embedded tissues

Introduction Canine visceral hemangiosarcoma (HSA) is a highly aggressive malignancy of endothelial cells with a poor prognosis and limited diagnostic tools. MicroRNAs (miRNAs) are small non-coding RNAs with emerging utility as diagnostic and prognostic biomarkers due to their stability and disease-specific expression profiles. Methods This study aimed to identify miRNA biomarkers for canine splenic and cardiac HSA using small RNA sequencing and quantitative PCR. Formalin-fixed, paraffin-embedded (FFPE) tissues were analyzed from 24 dogs with histologically confirmed HSA (18 splenic, six cardiac) and 12 non-neoplastic controls (six cardiac and six splenic). Results A total of 67 and 71 miRNAs were differentially expressed (DE) in splenic and cardiac HSA, respectively, with 18 miRNAs shared between both tumor types. Forty candidate miRNAs were selected for validation by RT-qPCR using customized panels. Thirteen miRNAs were validated as DE in each tissue type, showing strong concordance with sequencing results. Pathway enrichment analysis of validated miRNAs revealed a significant involvement in oncogenic signaling pathways, including the PI3K-Akt, MAPK, HIF-1, and Ras pathways. Discussion These results highlight miRNA signatures that may have diagnostic value in visceral HSA and support their use as biomarkers in archived tissues, with potential future application in liquid biopsy approaches.