Chronic inflammation and synovial hyperplasia are defining characteristics of rheumatoid arthritis (RA), an inflammatory condition that can ultimately lead to joint degeneration and functional impairment. Recent studies have uncovered a complex interplay between aging and RA3. Aging exacerbates the onset and progression of RA through mechanisms such as immune system dysregulation, chronic inflammation, cellular senescence, and metabolic disturbances4. Conversely, the adverse effects associated with chronic inflammation and RA treatments may also accelerate the aging process. The objective of this study was to identify potential senescence biomarkers for RA and to investigate the roles and mechanisms of senescence-related genes as well as immune infiltration in RA synovial tissues. This research aims to provide new insights into the underlying causes of RA, particularly in its early stages.
After integrating the three datasets retrieved from the GEO database, a total of fifty ARDEGs were identified through differential expression analysis. These ARDEGs exhibited significant enrichment in molecular functions related to DNA-binding transcription activator activity and RNA polymerase II specificity. Additionally, KEGG pathway analysis revealed substantial involvement in the PI3K-Akt signaling pathway. These results align with previous studies5,6,7,8. Notably, due to the extensive gene interaction data available in the STRING database and the sensitivity of the MCC algorithm to key nodes within networks, importing these 50 ARDEGs into STRING ultimately increased the number of identified key genes to 95. This finding underscores the importance of incorporating topological properties in the analysis of biological networks9, potentially revealing broader biological processes pertinent to disease research. The MCC algorithm, along with three machine learning screenings utilizing Cytohubba—a Cytoscape plug-in—identified four key genes: STAT1, JUN, MYC, and EGFR. Furthermore, gene expression analysis demonstrated that synovial samples from patients with rheumatoid arthritis (RA) exhibited significantly elevated levels of STAT1 and markedly reduced levels of JUN, MYC, and EGFR.
STAT1 (Signal Transducer and Activator of Transcription 1) is a member of the STAT protein family, which plays a crucial role in cytokine signaling and has significant functions in immunological regulation, cell division, and growth10. Recent mechanistic studies have identified STAT1 as a key regulator that links immunosenescence with rheumatoid arthritis (RA) inflammation. First, STAT1 promotes chronic inflammation through hyperactivation of the IFN-γ/JAK-STAT pathway, which correlates with increased synovial levels of IL-6 and TNF-α (r = 0.65–0.71, p < 0.001)11. Second, STAT1 exacerbates immunosenescence by facilitating T-cell exhaustion—evidenced by upregulation of PD-1—and impairing macrophage polarization, as demonstrated in aging murine models10. While our bioinformatics approach robustly prioritized STAT1 (AUC = 0.94, Fig. 7b), we recognize that exclusive reliance on computational data constrains mechanistic insights. For example, the observed negative correlation between STAT1 and regulatory T cells (r = −0.58, p = 0.002) necessitates validation through flow cytometry or single-cell RNA sequencing to establish causality. Although the pro-inflammatory role of STAT1 in rheumatoid arthritis (RA) is well-documented12, its age-specific regulatory mechanisms in elderly RA patients remain inadequately explored. Our findings contribute to existing knowledge in two significant ways: (1) Age-Dependent Expression: We found that STAT1 expression was markedly elevated in elderly RA patients (> 60 years) compared to younger cohorts (p = 0.003, Fig. 7a), whereas EGFR exhibited an inverse trend. This observation suggests that aging may disrupt the equilibrium between STAT1 and EGFR, potentially exacerbating disease progression—an insight not previously reported13. (2) Immune Microenvironment Associations: A strong correlation was identified between STAT1 and M1 macrophage infiltration (r = 0.67, p < 0.001), which contrasts with earlier studies focusing primarily on its interaction with Th17 cells14. These discoveries highlight the unique role of STAT1 in elderly RA and lay a foundation for age-stratified therapeutic strategies. Our findings align with the experimental research conducted by Chen Lili et al., which highlighted the significance of STAT1 as a potential biomarker15.
The elevated expression of STAT1 in monocytes and activated T cells from patients with rheumatoid arthritis (RA) closely correlates with its function in the interferon signaling pathway. Previous studies have established that STAT1 acts as a critical transcription factor within the interferon-γ (IFN-γ) signaling cascade, exacerbating synovial inflammation by activating downstream pro-inflammatory cytokines such as TNF-α and IL-616. In this study, monocytes exhibiting high levels of STAT1 expression were significantly enriched in the IFN-γ response pathway, suggesting that STAT1 may facilitate the polarization of monocytes towards a pro-inflammatory phenotype, thereby contributing to the dysregulation of the local immune microenvironment in RA joints17. Furthermore, the upregulation of STAT1 expression in activated T cells may enhance Th1/Th17 differentiation and further intensify the autoimmune response18.
The AP-1 transcription factor family, which includes the c-Jun protein encoded by the JUN (Jun proto-oncogene, AP-1 transcription factor subunit) gene, is associated with synovitis and cellular aging. The activator protein-1 (AP-1) family of transcription factors plays a crucial role in cell proliferation, differentiation, and apoptosis; it encompasses the c-Jun protein derived from the JUN gene. Through its regulation of pro-inflammatory cytokine production—such as IL-1, IL-6, and TNF-α—c-Jun enhances inflammatory responses19. Furthermore, c-Jun promotes synovial fibroblast-like cell proliferation, leading to synovial hyperplasia and joint deterioration19. Joint injury is further aggravated when the JNK/c-Jun signaling pathway is activated; this activation stimulates both the growth of synovial cells and the synthesis of inflammatory mediators20. The MYC gene, also referred to as the MYC proto-oncogene and bHLH transcription factor, is a member of the MYC gene family, which includes other proto-oncogenes such as C-, N-, and L-MYC. MYC regulates the expression of various genes by encoding a protein that acts as a basic helix-loop-helix (bHLH)-leucine zipper (LZ) transcription factor21. Furthermore, MYC promotes the formation of synovial fibroblast-like cells (FLS) and their aberrant proliferation and invasive capabilities through the PI3K-Akt and MAPK signaling pathways22. A study conducted by Jiawei Yao et al. revealed that the expression levels of JUN and MYC are significantly elevated in the synovial tissues of individuals with osteoarthritis23. These two genes may serve as potential biomarkers for differentiating between osteoarthritis and rheumatoid arthritis.
Epidermal Growth Factor Receptor (EGFR) is a member of the HER/ErbB family, which regulates EGFR growth, and belongs to the Receptor Tyrosine Kinase (RTK) family. The ErbB family plays a crucial role in controlling cell growth, survival, differentiation, and proliferation. One of the primary contributors to synovial tissue hyperplasia and inflammation in rheumatoid arthritis (RA) joints is the hyperactivation of the EGFR signaling pathway within RA synovial fibroblast-like cells (FLS)24. Notably, while this investigation observed a down-regulation of EGFR expression, numerous studies have reported an up-regulation of EGFR in RA13,25,26, necessitating confirmation through population-based cohorts. Furthermore, it has been demonstrated that EGFR enhances abnormal proliferation and invasive behavior of synoviocytes by activating both the PI3K-Akt and MAPK pathways22. Abnormal activation of EGFR exacerbates arthropathy in elderly patients with RA27. Although this gene test is currently employed in the treatment of cancer, its application in the management of rheumatoid arthritis (RA) remains infrequent. Nonetheless, it holds significant potential as a therapeutic target for older patients with RA.
Activated B cells, activated CD4 T cells, activated CD8 T cells, CD56dim natural killer (NK) cells, macrophages, and type 17 helper T cells (Th17) were identified as significantly up-regulated immune cell populations in rheumatoid arthritis (RA) through immune infiltration analysis. Conversely, natural killer cells, plasma-like dendritic cells (pDCs), follicular helper T cells (Tfh), and type 2 helper T cells (Th2) exhibited significant down-regulation in RA. The marked upregulation of activated T cells (CD4/CD8), B cells, macrophages, and Th17 suggests that patients with RA experience a robust pro-inflammatory immune response. This observation is consistent with findings from previous studies28,29. Th17 cells are well-recognized contributors to autoimmune inflammation and likely play a pivotal role in the pathophysiology of the disease. Furthermore, the diminished intra-immune homeostasis observed in RA patients is underscored by the down-regulation of regulatory and suppressive immune cell types such as NK cells and plasmacytoid dendritic cells30,31, which may exacerbate inflammation and accelerate disease progression.
Significant relationships between various immune cell types and the genes STAT1, EGFR, JUN, and MYC were identified in this study through Spearman correlation analysis. While EGFR, JUN, and MYC predominantly exhibited negative correlations with T cells and B cells, STAT1 demonstrated a positive correlation with these cell types. The favorable association between T cells and B cells with STAT1—a key regulator of the interferon signaling pathway—may underscore its critical role in enhancing immunological responses32. In addition to its involvement in monocyte and lymphocyte differentiation, STAT1 has been shown to positively regulate cytokine production, thereby improving adaptive immune responses33,34. Moreover, the immunomodulatory function of STAT1 has been validated across several diseases, further supporting the findings of the present study10,35,36,37. The majority of immune cell types, particularly T and B cells, exhibit a negative correlation with EGFR. Research has demonstrated that EGFR mutations can facilitate immune escape by triggering the PD-1/PD-L1 pathway38. Additionally, EGFR may influence the immune microenvironment in non-tumor contexts by suppressing T cell activity or altering the polarization status of macrophages39. The identification of this inverse relationship suggests that EGFR may play a significant immunomodulatory role in non-tumor disorders. Both JUN and MYC show a positive association with macrophages while exhibiting a negative correlation with T and B cells. This dual function in the inflammatory response may be indicative of their roles. JUN and MYC could either sustain the inflammatory environment through enhanced macrophage activity40 or promote inflammation by inhibiting adaptive immune responses41. Furthermore, MYC is recognized as a crucial downstream molecule within the AKT signaling pathway, which may influence immune responses in both tumor and non-tumor conditions42.
Naturally, this study has several limitations. First, the majority of the data were derived from public sources in the United States, necessitating further research that incorporates clinical data. Second, there is no experimental validation for this study; it relies solely on bioinformatics analysis. Future investigations should employ in vivo and ex vivo studies to elucidate the true roles of these genes in specific diseases and their potential therapeutic benefits. Third, the sample size utilized in this study was insufficient; to enhance its reliability moving forward, an increase in sample size is essential. A fourth significant limitation pertains to the use of distinct disease and senescence samples, which excluded individuals with rheumatoid arthritis (RA) as well as those suffering from debilitating conditions. Given that debility often coexists with older RA patients and may interact significantly to influence disease symptoms, treatment responses, and prognosis, this design could affect the generalizability and applicability of the findings. Therefore, future research should consider integrating these two patient sample types to explore potential biomarkers and therapeutic targets while also providing a more comprehensive evaluation of the relationship between aging and RA.
Synovial senescence may be closely associated with immunoinflammation, as suggested by this study’s preliminary investigation into the potential mechanisms involving senescence-related genes in rheumatoid arthritis (RA) synovial tissues. Furthermore, the four core genes identified may serve as novel targets for the diagnosis and treatment of RA due to their remarkable diagnostic capabilities. However, further experimental research is necessary to validate our findings.
