Identification and bioinformatics analysis of lncRNAs in serum of patients with ankylosing spondylitis

Ankylosing spondylitis (AS) is a common chronic inflammatory disease with an autoimmune etiology [1]. AS often affects the axial joints, including the sacroiliac joint and spine, and induces new bone formation and ultimately ankylosis [2, 3]. In most cases, the initial stage of AS is relatively insidious, which makes an early diagnosis difficult and leads to delayed treatment [4]. Although HLA-B27 misfolding [5], certain cytokines [6], and autophagy [7] are associated with AS progression, the etiology and pathogenesis of AS remain largely unclear. A better understanding of the underlying factors will help us to better understand the origins and progression of AS, and will help us identify new biomarkers or therapeutic targets.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that lack the protein-coding capacity, but can post-transcriptionally regulate the expression of certain genes [8, 9]. In recent years, some important lncRNAs have been observed in AS, which may be involved in AS pathogenesis. For example, MEG3 was found to sponge miR-146a and aberrant MEG3 downregulation could promote the inflammatory response through increasing the abundance of miR-146a [10]. Another study revealed that downregulation of LOC645166 promotes the NF-κB signaling via reduced blocking recruitment of the IKK complex to K63-linked polyubiquitin chains [11]. H19 is significantly upregulated in AS and mediates the inflammatory processes by acting as a competing endogenous RNA (ceRNA) in the miR22-5P-VDR-IL-17A/IL-23 axis in peripheral blood mononuclear cells [12]. Although several lncRNAs play important roles in diverse biological processes, the function and biological relevance of the vast majority of lncRNAs remain unclear. To the best of our knowledge, the expression profiles of lncRNAs, along with their potential biological functions, in the peripheral serum of patients diagnosed with AS still unclear in the current academic literature.

In the present study, we aimed to identify novel DElncRNAs for research and therapeutic purposes, and explored the signaling pathways underlying the DElncRNAs. To this end, we performed high-throughput RNA sequencing and bioinformatics analyses to identify the lncRNA expression profiles. Furthermore, the relevant pathways and biological functions of these RNAs were investigated using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome analyses. Finally, a regulatory network was constructed based on the identified lncRNAs.

Ankylosing spondylitis (AS) is a multifaceted ailment encompassing numerous factors [22]. Various treatment approaches have been devised to address AS; nevertheless, the majority of therapeutic interventions have proven inadequate in attaining desirable results. Furthermore, AS seriously affects the physical and mental health and quality of life of patients. Consequently, the timely identification and prompt initiation of treatment are imperative. The importance of lncRNAs in various processes, such as transcriptional, post-transcriptional, and epigenetic regulation, is well known [23]. Some lncRNAs are key determinants of metabolic activity, development, evolution, and disease pathophysiology [24]. Moreover, a growing body of evidence suggests that the dysregulation of lncRNAs plays an important role in the occurrence and progression of multiple immune-mediated inflammatory diseases, including AS [25]. However, only a small proportion of lncRNAs implicated in the AS pathogenesis has been identified, and no studies have investigated the lncRNAs present in the serum of AS patients. Hence, we conducted the present study to investigate the lncRNA expression profiles in the serum of AS patients, which may help to understand the pathophysiology and develop early diagnostic techniques and treatments for AS.

In our study, PCA plot and heatmap plot analyses illustrated that lncRNA expression profiles could distinguish AS patients from NC. Furthermore, the volcano plot showed that there were 145 DElncRNAs in AS patients compared to NC; among them, 72 lncRNAs were downregulated and 73 were upregulated. To explore the functional roles of the differentially expressed genes, we conducted GO, KEGG, and Reactome pathway analyses based on cis-regulation genes of DElncRNAs. The results indicate that DElncRNAs primarily participate in the regulation of protein ubiquitination, MHC class I-mediated antigen processing and presentation, MAP kinase activation, and interleukin-17 (Th17) signaling pathways, suggesting that DElncRNAs were enriched in pathways related to immune and inflammatory responses. The physiological function of the MHC class I molecules is to present antigenic or mutated peptides to the antigen receptors of CD8 + T cells and engage the receptors of natural killer cells for broad immunosurveillance against many pathogenic conditions [26, 27]. In line with this, a number of studies have reported a strong association between MHC class I allele human leukocyte antigen B27 and AS [5]. Based on previous evidence, the MAPK signaling pathway was implicated in the development of Th1 cells and macrophage activation [28]. Lin et al. found miR-134-5p inhibits osteoclastogenesis through the miR-134-5p/Itgb1/MAPK pathway [29]. Moreover, Pedersen et al. demonstrated that IL-17 is involved in the pathological mechanism of AS [30], especially in the occurrence and development of enthesitis, bone erosions, and bone formation [31, 32]. Some of the identified pathways, including the regulation of protein ubiquitination and signaling by NTRK1 (TRKA), have not been studied previously in the context of AS and thus warrant further investigation.

We validated the differential expression of four up- and downregulated highest fold-change lncRNAs via qRT-PCR to confirm the reliability of our RNA-seq data. This approach confirmed that six of the lncRNAs were differentially expressed in the expected directions (all p < 0.05). However, the levels of lnc-MRPS30-7:1 (p = 0.29) and lnc-FNTB-4:2 (p = 0.08) were not in line with the sequencing results. To determine the potential downstream targets of lncRNAs, we used the two most significantly upregulated lncRNAs (MALAT1 and NBR2) to construct a ceRNA. We found that MALAT1 may regulate the AS pathogenesis through the MALAT1/miR-1-3p/PPARG axis. Previous studies have shown that miR-1-3p is closely related to the pathogenesis of autoimmune diseases as well as several other diseases. As an example, the expression levels of MALAT1 and miR-1-3p were significantly higher in newly diagnosed patients with rheumatoid arthritis than NC [33]. Some studies have also shown that overexpression of miR-1-3p might facilitate Th17 differentiation by inhibiting the expression of ETS1 in naïve CD4⁺ T cells [34]. Peroxisome proliferator activated receptor gamma (PPARG) is a part of the subfamily of peroxisome proliferators activated receptors, which also includes PPARα and PPARβ/δ. This subfamily of receptors, especially PPARG, inhibits the key pro-inflammatory genes, such as NF-kB and TNFα, and the interleukins IL-1a and IL-6 [35]. In a study by Lin et al., lipocalin 2, which was regulated by PPARG, was found to be a potential pathway involved in concurrent ankylosis and inflammation in AS and inflammatory bowel diseases [36]. An additional study found that PPARG was highly expressed in peripheral serum of AS patients, which was validated by qRT-PCR [37]. Therefore, we speculate that there is a potential regulatory relationship between PPARG and MALAT1 in the serum of patients with AS.

MALAT1 is a large (6.5 kb), nuclear-enriched, and highly conserved lncRNA, which has important regulatory functions related to inflammation and is associated with autoimmune diseases. Increasing evidence indicates that MALAT1 acts as a competing endogenous RNA (ceRNA) to reduce the repression effect of miRNA on the expression of certain inflammatory factors [38]. Zhou et al. suggested that the downregulation of lncRNA MALAT1 hindered the inflammation of microglial cells and prevented the progression of acute spinal cord injury via the modulation of the miR-199b/IKKβ/NF-κB signaling pathway [39]. Pathological bone formation is another pivotal mechanism involved in the pathology of AS. In vitro knockdown of MALAT1 suppresses the proliferation of the human osteoblast cell line hFOB 1.19 [40], and MALAT1 sponging of the microRNA miR-30 promotes the osteoblastic differentiation of mesenchymal stem cells by inducing RUNX2 expression [41]. Furthermore, the downregulation of lncRNA MALAT1 decreased miR-558-mediated GSDMD expression to enhance cell viability and suppress apoptosis and pyroptosis of chondrocytes in AS [42]. Therefore, MALAT1 plays a unique role in the AS pathogenesis, although additional research is needed to understand the mechanisms whereby the candidate lncRNAs impact disease progression.

Our results provide new insights into the underlying mechanisms of AS. However, some limitations of our study should be considered. First, the sample size was relatively small, and all cases were recruited from a single center. Therefore, large-scale, multi-center cohort studies are needed to confirm the accuracy of our RNA-seq results. Second, follow-up studies using in vitro experiments and animal models are required to determine the underlying regulatory mechanisms of the DElncRNAs in AS pathogenesis.