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.