FOXC1 and its lncRNA FOXCUT link DNA methylation to a chondrocyte phenotype dysfunction

Abstract Objective With current osteoarthritis (OA) therapies limited to symptom management, understanding molecular drivers of disease progression is critical. As epigenetic mechanisms increasingly appear central to OA pathogenesis, this study aimed to identify epigenetically dysregulated targets and to delineate the functional contribution of FOXC1 and its lncRNA FOXCUT to chondrocyte behavior and extracellular matrix (ECM) integrity. Methods Chondrocytes and bone marrow-derived stem cells (BMSCs) were isolated from OA patients and healthy donors. Genome-wide DNA methylation profiling and integrative bioinformatic analyses were performed to identify differentially methylated regulatory elements. Functional validation of FOXC1 and FOXCUT was carried out through gene silencing in a cartilage-on-a-chip (OoC) platform. RNAscope in situ hybridization was used to determine spatial expression patterns within a near-native joint microenvironment. Additional confirmation was obtained in 3D chondrocyte pellet cultures to evaluate ECM organization and collagen type II production following gene knockdown. Results FOXC1 and FOXCUT were identified as hypomethylated and upregulated in OA chondrocytes and BMSCs. Silencing of either gene in the OoC model altered chondrocyte morphology, with FOXC1 knockdown producing the most marked phenotypic changes. RNAscope analysis revealed distinct spatial expression of FOXC1 and FOXCUT consistent with their regulatory roles. In 3D pellets, knockdown of FOXC1 or FOXCUT affected ECM organization and reduced collagen type II deposition. Conclusions This study provides the first functional evidence linking epigenetic dysregulation of FOXC1 and FOXCUT to OA pathogenesis. Their roles in modulating chondrocyte phenotype and ECM integrity highlight them as promising biomarkers and candidates for future epigenetic-based therapeutic strategies in OA.