miR-424-5p Regulates Stem-like and Malignant Phenotypes in Osteosarcoma by Targeting FZD4

Background/Objectives: OS is characterized by marked tumor heterogeneity, stemness-associated phenotypes, metastatic potential, and poor prognosis. FZD4 is an important Wnt signaling receptor involved in stem cell regulation and tumor progression, but its role and microRNA-mediated regulation in OS remain unclear. The aim of this study was to investigate the relationship between FZD4 expression, OS stemness, malignant progression, and upstream microRNA regulation. Methods:Human OS single-cell RNA sequencing data were analyzed using Seurat, CytoTRACE, and Monocle2. Tumor cell subpopulations, developmental potential, and pseudotime trajectories were evaluated. Functional enrichment analyses were performed to identify pathways associated with high FZD4 expression. MicroRNA-mRNA interaction analysis was used to predict upstream regulatory microRNAs, and immunohistochemistry, Western blotting, and in vitro and in vivo experiments were performed for validation. Results: FZD4 was mainly enriched in OS tumor cell subpopulations and was significantly associated with poor prognosis. Tumor cells with high FZD4 expression showed increased developmental potential, stemness features, and enrichment in early developmental states. Functional enrichment analysis indicated activation of epithelial-mesenchymal transition, angiogenesis, inflammatory response, KRAS signaling, hypoxia, glycolysis, and Wnt-related pathways. Clinical validation confirmed elevated expression of FZD4, OCT4, and SOX2 in OS tissues. FZD4 knockdown inhibited OS cell stem-like phenotypes and malignant behaviors, proliferation, and migration, while miR-424-5p suppressed these malignant phenotypes by targeting FZD4. Importantly, rescue experiments demonstrated that restoration of FZD4 substantially reversed the inhibitory effects of miR-424-5p mimics on sphere formation, migration/invasion, proliferation, and stemness-associated cell populations. Conclusions: Our findings identify miR-424-5p as an upstream suppressor of FZD4 and demonstrate that the miR-424-5p/FZD4 axis regulates stem-like and malignant phenotypes of OS cells, at least partly through Wnt/β-catenin signaling. This axis may represent a potential therapeutic target for OS.