INTRODUCTION: Osteosarcoma, a highly aggressive bone cancer primarily affecting children and young adults, remains a significant challenge in clinical oncology. Metastasis stands as the primary cause of mortality in osteosarcoma patients. However, the mechanisms driving this process remain incompletely understood. Clarifying the molecular pathways involved in metastasis is essential for enhancing patient prognoses and facilitating the development of targeted therapeutic strategies. METHODS: RNA sequencing (RNA-Seq) analysis was employed to compare three conditions, hFOB1.19 versus Saos-2, hFOB1.19 versus SJSA-1, and Saos-2 versus SJSA-1, involving non-cancer osteoblasts (hFOB1.19) and highly metastatic osteosarcoma cell lines (Saos-2 and SJSA-1). Additionally, ENA datasets of RNA-Seq from osteosarcoma biopsies were included. Differentially expressed genes (DEGs) were identified and analyzed through enrichment pathway analysis and protein-protein interaction (PPI) networks. Additionally, for gene candidates, a biochemical evaluation was performed. RESULTS: DEGs associated with biological functions pertinent to migration, invasion, and metastasis in osteosarcoma were identified. Notably, matrix metalloproteinase-2 (MMP-2) emerged as a promising candidate. Both canonical or full-length (FL-mmp-2) and N-terminal truncated (NTT-mmp-2) isoforms were discerned in biopsies. Moreover, MMP-2's activity was characterized in cell lines. Additionally, mRNA expression of voltage-gated sodium channels (Na CONCLUSIONS: The gene functional enrichment analysis of DEGs enabled the identification of interaction networks in osteosarcoma, thereby revealing potential biomarkers. Moreover, the elucidated co-participation of TTX-sensitive Na