Discussion
To date, tens of thousands of circRNAs have been reported in eukaryotic species, and their functional importance has attracted increasing interest in recent years[24, 25]. In a recent study, over 140,790 circRNAs were profiled in 20 clinically relevant tissues, revealing the tissue-specific expression patterns of some of these human circRNAs[26]. The sequestration of miRNAs via the sponge-like ceRNA mechanism is one of the best-characterized functional mechanisms by which circRNAs influence cell biology[26]. Some circRNAs possess numerous miRNA binding sites that enable them to effectively suppress miRNA functionality[30, 31]. Clear evidence has demonstrated roles for circRNAs in proliferation[32, 33], cell cycle progression[34], apoptosis[35, 36], and autophagy[37, 38].
In this study, we analyzed the expression of circRNA in PBMCs from AS patients and identified 131 DECs (89 upregulated and 42 downregulated) compared to DECs isolated from healthy controls. To verify the reliability of the sequencing data, three up-regulated (hsa_circ_0000702, hsa_circ_0006209, hsa_circ_0047920) and three down-regulated (hsa_circ_0001543, hsa_circ_0072697, hsa_circ_0005076) circRNAs were selected for RT-qPCR-based validation when analyzing a larger cohort of samples from AS and CN patients. KEGG analyses of the target genes associated with the six circRNAs with putative ceRNA functionality revealed these genes to be related to the p53, Adherens junction, ErbB, cell cycle, B cell receptor signaling, neurotrophin, Toll-like receptor, T cell receptor, VEGF, mTOR, focal adhesion, adipocytokine, Notch, NOD-like receptor, FceRI, and apoptotic signaling pathways. Further, correlation and ROC curve analyses revealed the expressions of hsa_circ_0006209 and hsa_circ_0047920 to be positively correlated with hs-CRP levels in AS patients, while hsa_circ_0072697 levels were positively correlated with ESR and BASDAI levels in this same patient cohort. ROC curve analysis demonstrated that hsa_circ_0072697 had the highest potential as a diagnostic biomarker for AS among the six validated circRNAs. Moreover, the ceRNA network constructed based on these circRNAs offers valuable insights into the regulatory mechanisms underlying AS and could be utilized to identify potential therapeutic targets. Recent studies have shown that hsa_ CircRNA_ 1012732 is correlated with indicators of AS disease activity, hsa_ CircRNA_ 1001544, hsa_ Circle_ 0000652 may be a molecular marker for diagnosing AS[39, 40].
As part of homeostasis, proliferation, inflammation, and apoptosis are highly regulated cellular processes[41]. Previous studies have shown that CircRNA is involved in various biological processes, including cell proliferation, apoptosis, invasion, and migration[42-45]. In the present study, in vitro cell experiments were used to thoroughly investigate the role of hsa_cir_0072697 in the pathogenesis of AS. It was found that overexpression of hsa_circ_0072697 inhibited PBMC proliferation and promoted apoptosis. Conversely, knockdown of hsa_circ_0072697 promoted PBMC proliferation and inhibited apoptosis. Additionally, hsa_circ_0072697 expression levels affected the cell cycle progression of PBMCs. We then analyzed the effect of hsa_circ_0072697 expression on inflammatory cytokine expression (TNF-α, IL-4, IL-10, and IL-17) and key genes in the NF-kB pathway. Our results showed that overexpression of hsa_circ_0072697 increased IL-4 and IL-10 concentrations and decreased IL-17 and TNF-α concentrations. It also led to decreased expression of hsa-miR-145-5p, MMP3, MMP9, and NF-kB p65 mRNA, and increased expression of IKBα. Conversely, knockdown of hsa_circ_0072697 resulted in the opposite effects. Moreover, IF assays confirmed the effects of different hsa_circ_0072697 expression levels on p-IKBα and p-NF-κB. Our study highlights the importance of hsa_circ_0072697 in regulating the expression of key genes involved in the pathogenesis of AS, especially those involved in the NF-κB pathway. These findings shed new light on the molecular mechanisms underlying AS development and suggest potential therapeutic targets for the disease. However, further studies are needed to fully understand the underlying mechanism of hsa_circ_0072697 in AS pathogenesis and its potential as a therapeutic target.
Several limitations of this study should be acknowledged. Firstly, the sample size is relatively small, and larger studies with multi-ethnic populations are needed to validate and extend our findings. Secondly, we did not assess the ability of the identified circRNAs to differentiate AS patients from those with other autoimmune diseases, such as SLE, MS, or RA. Future studies should aim to evaluate the specificity of these biomarkers by comparing their ability to distinguish AS from other related conditions.
In summary, we employ a high-throughput RNA-seq method in PBMCs samples to identify specific circRNA biomarkers that are specifically associated with the incidence of AS patients. The identified DECs and related ceRNA functions provide new directions for future research regarding the mechanistic basis for the pathogenesis of AS. Specially, we confirm that hsa_circ_0072697 has a good diagnostic value in AS patients, and it could suppress the progression of AS by targeting NF-κB pathway. These findings shed new lights on AS mechanism and clinical application in patients.