Conclusion
Our findings highlight the critical role of CDKN1A in promoting cartilage formation in both in vivo and in vitro and suggest its potential as a therapeutic target for OA treatment.
Objective
Chondrocyte death is the hallmark of cartilage degeneration during osteoarthritis (OA). However, the specific pathogenesis of cell death in OA chondrocytes has not been elucidated. This study aims to validate the role of CDKN1A, a key programmed cell death (PCD)-related gene, in chondrogenic differentiation using a combination of single-cell and bulk sequencing approaches. Design: OA-related RNA-seq data (GSE114007, GSE55235, GSE152805) were downloaded from Gene Expression Omnibus database. PCD-related genes were obtained from GeneCards database. RNA-seq was performed to annotate the cell types in OA and control samples. Differentially expressed genes (DEGs) among those cell types (scRNA-DEGs) were screened. A nomogram of OA was constructed based on the featured genes, and potential drugs targeting the featured genes were predicted. The presence of key genes was confirmed using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), Western blot (WB), and immunohistochemistry (IHC). Micromass culture and Alcian blue staining were used to determine the effect of CDKN1A on chondrogenesis.
Results
Six cell types, namely HomC, HTC, RepC, preFC, FC, and RegC, were annotated in scRNA-seq data. Five featured genes (JUN, CDKN1A, HMGB2, DDIT3, and DDIT4) were screened by multiple biological information analysis methods. TAXOTERE had the highest ability to dock with DDIT3. Functional analysis indicated that CDKN1A was enriched in processes related to collagen catabolism and acts as a positive regulator of autophagy. Additionally, CDKN1A was found to be associated with several KEGG pathways, including those involved in acute myeloid leukemia and autoimmune thyroid disease. CDKN1A was confirmed down-regulated in the joint tissues of OA mouse model and OA model cell. Inhibiting the expression of CDKN1A can significantly suppress the differentiation of OA chondrocytes.