Pos0699 Rna Sequencing Reveals Crucial New Players in Osteoarthritis Cartilage Calcification

Background: Osteoarthritis (OA) is a degenerative joint disease characterized by pathologic calcification of cartilage, where chondrocytes deposit calcium-containing crystals. Understanding the precise mechanisms behind calcification is challenging but crucial for developing effective therapeutic strategies. Objectives: This study aims to identify key players in knee OA cartilage calcification, providing insights for targeted interventions. Methods: Primary murine chondrocytes were stimulated with secondary calciprotein particles (CPP2) or left untreated (NT) in DMEM high glucose for 6 hours. Lysates were prepared according to MERCURIUS™ Extraction-free DRUG-seq kit and submitted to Alithea Genomics SA (Lausanne, Switzerland) for Bulk RNA sequencing on an Illumina Novaseq 6000. STARsolo v2.7.9a aligned and quantified reads against the mouse (GRCm38.102) genome. Differentially expressed (DE) genes were identified with DESeq2 (abs(LogFC)>1, FDR < 0.05), and the top 50 DE genes were cross-referenced with human OA datasets from previous studies (i.e. OA vs healthy cartilage, or damaged vs undamaged cartilage). Finally, human primary chondrocytes from 5 osteoarthritis patients were stimulated with CPP2 to validate gene modulation by qRT-PCR. Results: We first confirmed that CPP2 induce calcification in primary murine chondrocytes (Figure panel A). Bulk RNA sequencing revealed 1466 differentially expressed genes (668 up- and 798 downregulated) between CPP and NT murine chondrocytes. Out of the top 50 DE genes (Figure panel B), 18 were significantly upregulated and 4 downregulated in at least 1/7 OA cartilage datasets. Interesintgly, previously studied genes in osteoarthritis (ERRFI1, NGF, INHBA, RCAN1, and GREM1) were found and new candidates (S100A10, EIF5A, and TNFRSF12A) emerged. In primary human chondrocytes, we confirmed upregulation of NGF, INHBA, RCAN1 and TNFSFR12A by CPP2, and downregulation of COL9A1 (Figure panel C). Conclusion: Our study unveils NGF, INHBA, RCAN1 and TNFSFR12A as key players in cartilage calcification, presenting promising avenues for therapeutic interventions in an area where effective treatments are currently limited. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.