Osteoarthritis (OA) is a condition that involves movable joints and features cellular stress and ECM degradation attributed to micro- and macro-injury, which activates maladaptive repair responses including pro-inflammatory pathways of innate immunity. OA first occurs as a molecular derangement (metabolic abnormality in joint tissue) and subsequently as anatomic and/or physiologic derangements (characterized by cartilage degradation, bone remodeling, osteophyte formation, joint inflammation, and loss of normal joint function) and eventually results in illness.1 Traditionally, it is perceived that cartilage is first afflicted by early OA. Under physiological stress, chondrocytes are responsible for maintaining the balance between cartilage matrix degradation and synthesis. When chondrocytes are overloaded by injury or stress, the rate of matrix degradation exceeds the rate of synthesis, which induces joint tissue degradation and culminates in OA.19 Researchers presume that OA is initiated by changes in the subchondral bone.20 In OA, alterations in the subchondral bone mainly include increases in subchondral bone volume fraction, trabecular thickness and connectivity density of the subchondral bone due to loss of cartilage, which promotes bone remodeling and results in osteophyte formation.21 OA also involves the innervated synovial tissue, a potential source of peripheral pain in OA; knee joints with painful OA are shown to largely enriched with gene-encoding neuronal proteins that promote neuronal survival under cellular stress, participate in calcium-dependent exocytosis in synaptic terminals and regulate GABA (γ-aminobutyric acid)-ergic activity.22 Epidemiological studies prove that OA is an intricate disorder involving numerous factors.23 In terms of OA diagnosis, clinical approaches remain the mainstay in spite of the extensive use of imaging techniques; as to OA treatment, conservative interventions are demonstrated to have limited efficacy; patients with advanced OA usually have no other options besides surgery. All this unravels the profound significance and excellent prospect of research and studies on the prevention and early diagnosis and treatment of OA.5 The extraordinary growth of bioinformatics enables big data analysis of genes and proteins to facilitate the identification of potential diagnostic and therapeutic targets in oncology and other research fields.8 However, bioinformatics tools are seldom used in OA studies. This study aimed to explore the latest biological information that emerges with the onset of OA and identify new candidate biomarkers by using bioinformatics tools for big data analysis of OA-related molecular expression, thereby offering reference to the discovery of potential targets for the early diagnosis and treatment of OA, as well as experimental molecules for OA studies.
Based on the high-throughput sequencing dataset obtained from the GEO database, three R packages, including DESeq2, EdgeR, and Limma, were utilized to identify DEGs, respectively. Eventually, 117 downregulated and 174 upregulated overlapping DEGs were identified. The GO analysis revealed that the DEGs were mostly enriched in two categories of biological processes: (1) ECM degradation and collagen breakdown; (2) development, ossification and differentiation of cells (chondrocytes) and cellular responses to hormones, nutrient levels, oxygen levels, external stimulus, and starvation.
ECM status changes, damage and degradation have been accepted as basic pathological changes in OA.24 Cartilage does not contain blood vessels but a dense ECM with a sparse distribution of chondrocytes.25 Accounting for 1-5% of the total cartilage tissue volume, chondrocytes provide powerful support for the synthesis of ECM proteins such as collagen, hyaluronic acid or glycoprotein and proteoglycan, with the collagen level contributing to approximately 60% of the dry weight of cartilage.26,27 ECM degrading enzymes (metalloproteinases, i.e., ADAMTSs) and matrix metallo proteinases (MMPs) can induce ECM degradation, which is considered as a characteristic manifestation of OA.28 Hyaline cartilage can be precursory or permanent. Healthy cartilage that exists in articular joints is defined as permanent cartilage, which consists of resting chondrocytes; under normal conditions, resting chondrocytes have a low proliferation rate and do not undergo terminal differentiation or endochondral ossification (EO).29 However, in the presence of certain diseases, some articular chondrocytes are likely to lose their normal phenotypes and EO-like proliferation.30 EO occurs when chondrocytes undergo active proliferation and produce a cascade of cells, with some being enlarged and others growing into hypertrophic chondrocytes through hypertrophic changes. As these cells undergo a dramatic increase in their volumes, the surrounding is mineralized to develop bone tissue.31 In the meantime, cartilage is hardened due to alteration in its elastic nature through calcification, making it increasingly difficult for chondrocytes to absorb nutrients. This leads to apoptosis of most chondrocytes and formation of small cavities within the tissue, leaving sufficient room for blood vessel invasion in the hardened structure and resulting in a shift from cartilage towards trabecular bone. Numerous studies have shown that the highlight events that occur in EO are also observed in OA, such as chondrocyte proliferation, hypertrophic differentiation of chondrocytes, cell death, calcification or mineralization, blood vessel invasion, and chondrocyte apoptosis.28,29,30 Particularly, hypertrophic differentiation of chondrocytes is considered as one of the major pathological changes in OA.32 Hypertrophy generally refers to an increase in the size and volume of cells. Hypertrophic chondrocytes start to express osteogenic differentiation-related genes and produce mineralized ECM proteins.33,34 Besides, hypertrophic cells undergo decreases in hyaline cartilage markers, such as aggrecan, collagen type II, and SOX9. Despite the essential role of hypertrophic changes in chondrocytes in bone growth and development, this mechanism is a double-edged sword in the presence of disease.35 Although chondrocyte clarification is known as the final stage of chondrocyte hypertrophy, it remains unclear whether hypertrophic chondrocytes are able to fully differentiate into bone cells. Despite all that, apoptosis of hypertrophic chondrocytes and lacunar emptying within OA cartilage are reported to give rise to the loss of articular cartilage and contribute to the generation of osteophytes by subchondral bone, that is, cartilage tissue is gradually replaced by bone tissue during the EO-like process in OA.28, 29, 36 Additionally, chondrocyte senescence is suggested to play a crucial role in the pathological process of OA.37 Cellular senescence may result from external stimuli or stress, oxidative stress, DNA damage, telomere shortening, oncogene activation and other factors.38 It is found that mechanical stress stimuli increase oxidative stress to accelerate the progression of chondrocyte senescence in vitro.39 Cellular responses to oxygen levels and external stimuli as shown in the GO analysis are consistent with these opinions.
The results of our KEGG enrichment analysis also support these opinions. The PI3K/AKT/mTOR signaling pathway has a complex structure known to involve over 150 proteins40 and play an essential role in many cellular processes to maintain homeostasis, such as cell cycles and cell survival, inflammation, metabolism and apoptosis.41 Many studies have shown that the PI3K/AKT/mTOR signaling pathway is strongly associated with cartilage degradation, synovial inflammation, and subchondral bone sclerosis in OA.42 Chondrocytes are suggested to exhibit two different mechanisms of senescence: (1) replicative senescence, and (2) stress-induced premature senescence. Notably, the P53 signaling pathway that was shown to be enriched with upregulated genes in the KEGG enrichment analysis is thought to play a role in the replicative senescence and apoptosis of chondrocytes.43 In addition to chondrocytes, synovioblasts are also suggested to accelerate the progression of OA through senescence. When exposed to H₂O₂ or TNF-α, synovioblasts exhibit increased senescence and promote mRNA expression and protein secretion in IL6, CXCL8, CCL2 and MMP3,44 with the TNF signaling pathway being included in the KEGG enrichment results. Likewise, the FoxO signaling pathway present in the KEGG enrichment results is considered as a critical signaling pathway in OA. FoxO1, FoxO3, FoxO4, and FoxO6 belong to the family of FoxO transcription factors that play a crucial role in the development and aging processes.45 It is found in a study that in OA chondrocytes, inflammatory mediators and cartilage-degrading enzymes are reduced due to overexpression of FoxO1, which demonstrates the critical role of FoxOs in cartilage development, maturation and homeostasis as well as the protective properties of FoxOs against OA-associated cartilage damage.46
Following the analysis of DEGs based on the PPI network, MCODE was used for the identification of hubgenes, which yielded a total of 23 highly expressed hubgenes. These genes were ranked by MCODE score and verified by the other two datasets. Finally, 11 out of the 23 genes were found to exhibit differential expression in those datasets. The two datasets used for verification have a limited number of registered samples, which may bring a bias to this study and fail to reflect gene expression accurately. Particularly, because GSE169077 contains only 5 normal samples and 6 OA samples, the differences in gene expression were shown to lack statistical significance. Despite all that, we still spotted its value for analysis. KIF20A, also known as RAB6KIFL, is a kinesin family member involved in such cellular processes as mitosis, migration and intracellular transport and essential to cell division.47 Currently, most KIF20A studies focus on oncology. Overexpression of KIF20A is found in melanoma, bladder cancer, and breast cancer;48,49,50 migration and invasion of pancreatic cancer cells are inhibited by downregulating the expression of KIF20A.51 Ribonucleotide reductase (RNR) is an enzyme that participates in cell cycles, which contains two subunits, including the regulatory subunit RRM1 and the catalytic subunit RRM2 involved in DNA replication and repair.52 Aberrantly upregulated expression of RRM2 promotes rapid cell division through an increased accumulation of dNTPs,53 which is closely associated with many types of cancer. However, the role of KIF20A or RRM2 has not been reported. Since chondrocytes have a relatively low proliferation rate, it is puzzling that KIF20A and RRM2, which have a strong association with cell division, are highly expressed in OA chondrocytes; further studies are needed to clarify the mechanisms of action of these genes in OA chondrocytes and investigate whether they are potential therapeutic targets for OA treatment. CDK1 is a member of the cyclin-dependent kinase (CDK) family. Increased CDK activity induced by the alteration of DNA damage and mitotic checkpoints is demonstrated to drive cell cycles.54 If the activation of CDKs is out of control, this can lead to unexpected cell proliferation, as well as chromosomal and genomic instability.55 It is currently believed that CDK1 can serve as a potential therapeutic target for cancer treatment, which achieves therapeutic goals by gaining control over cell proliferation in lung or breast cancer.56,57 CEP55 (centrosomal protein 55 kDa) is dispensable for the whole cell cycle, especially in the stage of cytokinesis.58 Moreover, the CEP55 gene is found to induce breast cancer cell death in vitro during mitosis and sensitize breast cancer cells to antimitotic agents; CEP55-dependent antimitotic treatment leads to early activation of CDK1/Cyclin B and thereby initiates cell death from the G2/M phase.59 Meanwhile, through observation of the miRNA-mRNA regulatory networks, it was noted that miR-6838-5p might produce regulatory effects on CEP55 and CDK1 simultaneously. There are few published works in relation to miR-6838-5p and most are oncology studies, in which miR-6838-5p is found to inhibit the Wnt pathway by targeting WNT3A to suppress cell proliferation and migration in triple-negative breast cancer.60 Another similar report pointed out that by targeting GPRIN3 via the Wnt/β-Catenin signaling pathway, miR-6838-5p inhibited the malignant behaviors of gastric cancer cells, including cell growth, migration and invasion.61 Since the effects of CDK1, CEP55 and miR-6838-5p on the cellular processes of chondrocytes such as cell cycle, mitosis, and death are unclear, it is worthwhile to conduct further studies and explore their value as therapeutic targets.
This study has some limitations: (1) the analysis of a single dataset may produce biased results in spite of the superior data quality and application of different algorithms; (2) The verification of some molecules is based on two other datasets with a smaller sample size, especially GSE169077, which only contains 5 normal samples and 6 OA samples. Therefore, it is clear that the verification results will bring a bias to the present study. (3) Because this study is carried out on the basis of an online database, the results should be verified by experiments to draw more accurate conclusions.