Osteoarthritis (OA) is characterised by degeneration of the articular cartilage, which is primarily attributable to changes in chondrocyte viability, which in turn promotes a reduction in the rate of catabolism, thereby affecting chondrocyte structure [8–12]. HIF-2α (Epas1) promoter analysis has shown that nuclear factor (NF)-κB regulates HIF-2α transcriptional activity and that the NF-κB/HIF-2α signalling pathway is closely associated with the development of OA caused by articular cartilage damage . This highlights the importance of elucidating the mechanisms underlying HIF-2α over-expression in chondrocytes leading to chondrocyte damage and OA. In the present study, we used the GCBI analysis platform [14–18] for DEG, GO term and pathway enrichment, pathway network, and gene signal network analyses of the GEO gene profiling GSE104794 dataset. Subsequent intersection analysis of 104 hub genes identified in the gene signal network analysis of GSE104794 and 864 DEGs identified on the basis of the DEG analysis of GSE51588 yielded 10 key genes (LUM, ENTPD3, SMPD3, FGFR3, GPX3, IRAK3, EREG, HTR2A, TLR2, and CDA), which were verified by qRT-PCR in OA.
In this study, we identified 542 that were differentially expressed between the experimental and control groups, among which 418 and 124 were up- and downregulated respectively, under the conditions of HIF-2α over-expression. These 542 DEGs can be considered as those involved in the development and progression of OA in response to the over-expression of HIF-2α. The results of GO biological process analysis indicated that to a large extent, these DEGs play roles in the inflammatory response and cell apoptosis. Subsequent pathway analysis revealed that the 542 DEGs are significantly enriched in the cytokine–cytokine receptor interaction, pathways in cancer and rheumatoid arthritis, metabolic pathways, HIF-1 signalling pathway, legionellosis, Toll-like receptor signalling pathway, Chagas disease (American trypanosomiasis), MAPK signalling pathway, and pertussis. Among these enriched pathways, the MAPK signalling pathway is essential in signal transduction, integration, and amplification, and is a major component of various eukaryotic cellular processes [19, 20]. This pathway also regulates the over-expression of MMP13 induced by cytokine stimulation in response to mechanical injury and inflammation . MMP13 plays a major role in the development of OA and is considered a prominent biomarker reflecting the progression of arthritis and therapeutic efficacy [19, 22, 23].
On the basis of pathway network analysis, we identified 54 core pathways, including the MAPK signalling pathway, apoptosis, pathways in cancer and the cell cycle, p53 signalling pathway, focal adhesion, T cell receptor signalling pathway, glycolysis/gluconeogenesis, JAK-STAT signalling pathway, and cytokine–cytokine receptor interaction. These finding would thus tend to indicate that these pathways may play the most important roles in OA caused by HIF-2α over-expression. Further analysis of their interrelationships indicated that cancer-associated pathways may be relatively upstream pathways, given that the downstream pathways included the seven pathways MAPK signalling pathway, apoptosis, cell cycle, p53 signalling pathway, focal adhesion, JAK-STAT signalling pathway, and cytokine–cytokine receptor interaction. Cancer-associated pathways have been established to play roles in numerous diseases [24–28]; however, to the best of our knowledge, this is the first study to reveal that these pathways may also play a major role in OA.
To screen for key genes affected by the over-expression of HIF-2α in chondrocytes, we performed gene signal network analysis of the 542 DEGs, which revealed Nfkb1, Tlr2, Nt5e, Enpp1, Entpd3, Vegfa, Ptgs2, Socs3, Fos, and Epas1 to be the top 10 genes in the network. Among these, NFKB1 is an important component involved in the differentiation of osteoclasts. Consequently, inhibiting the expression of NFKB1 could represent an effective strategy for the treatment of a range of bone and joint diseases . Our findings in the present study indicate that the over-expression of HIF-2α in chondrocytes promotes the upregulated expression of NFKB1, which may play a major role in the development and progression of OA.
It has been established that subchondral bone tissue plays a key role in the pathogenesis of OA , and microarray analysis of knee subchondral bone samples from OA and non-OA patients could provide clues regarding the pathogenic mechanism of OA, thereby providing a basis for the development of novel diagnostic markers and therapeutic targets . Furthermore, it has been demonstrated that there is an interaction between articular cartilage and subchondral bone in OA. Consequently, it can be speculated that those genes that are differentially expressed in both cartilage and subchondral bone may serve as key genes in the development of OA . In the present study, we identified 10 key genes (LUM, ENTPD3, SMPD3, FGFR3, GPX3, IRAK3, EREG, HTR2A, TLR2, and CDA) based on an analysis of the intersection of 104 hub genes from GSE104794 (cartilage) and 864 DEGs from GSE51588 (subchondral bone) datasets, among which SMPD3, encoding a lipid-metabolising enzyme, has been identified as a key regulator of skeletal development, and its expression in chondrocytes and osteoblasts is required for normal endochondral bone development . Our findings in the present study indicate that SMPD3 may be one of the key genes implicated in the occurrence and development of OA.
This study did, nevertheless, have certain limitations. Notably, our results are all based on bioinformatic analysis, and therefore, require further validation with experimental and clinical data. Moreover, the sample size was relatively small, which may have introduced a degree bias in the results obtained.
In summary, the key genes (LUM, ENTPD3, SMPD3, FGFR3, GPX3, IRAK3, EREG, HTR2A, TLR2, and CDA) screened out based on bioinformatic analysis may serve as targets for novel intervention strategies for patients with osteoarthritis as well as potential molecular diagnostic markers for the prevention and treatment of this disease.