Comprehensive analysis of potential ceRNA network and immune cell infiltration in intervertebral disc degeneration

Lumbar degenerative disease (LDD) is a common disease in middle-aged and elderly people, with chronic low back pain as the main clinical manifestation. With the aging population, LDD has become a serious public health problem [1]. Intervertebral disc degeneration (IDD) is the pathological basis for its development [2], and a series of diseases such as lumbar disc herniation, endplate inflammation and lumbar spinal stenosis can develop as a result. IDD can occur in adolescence and gets progressively worse with age, with the prevalence of IDD exceeding 90% in people older than 50 years [3]. At present, the pathogenesis of IDD is still not specifically elucidated. Anti-inflammatory and analgesic drugs are often used in conservative treatment to relieve symptoms, and surgical treatment may still leave residual back and leg pain or generate new pain due to degeneration of adjacent segments. Therefore, it is helpful to understand the pathogenesis and find key targets for the treatment of IDD.

With the continuous development of biology, microRNAs (miRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs) have been shown to be involved in the occurrence and development of IDD and are considered to be the key to diagnosis and treatment. MiRNAs can bind to target mRNAs to destabilize them and inhibit mRNAs translation or cause their degradation, thus achieving post-transcriptional regulation of gene expression. MiRNAs play an important role in the biological process of IDD and participate in it by regulating inflammatory response, cell proliferation, cell apoptosis and other pathways [4, 5]. Dong found that miR-640 inhibited the expression of β-catenin and EP300, thereby suppressing WNT signalling and inducing nucleus pulposus cell degeneration [6]. Based on this, they proposed that miR-640 inhibitor therapy is a potential therapeutic target for low back pain. As research progressed, circRNAs were found to regulate gene expression by sponging miRNAs to achieve the purpose of regulating diseases [7]. Nowadays, more and more studies have shown that circRNAs also play an important role in IDD, participating in extracellular matrix metabolism, cellular senescence, apoptosis, inflammatory response and other processes [8]. Hu found that circRNA_0022382 ameliorated IDD by sponging miR-4726-5p to downregulate TGF-β3 expression, suggesting that circRNA_0022382 may serve as a new way to prevent and treat IDD [9]. The microenvironment of intervertebral disc also has an important impact on the development of IDD. Some miRNAs (miR-299-5p、miR-17), mRNAs (TGF-β、IL-1β) and lncRNA (SNHG5) have been reported to affect the development of IDD by mediating immune cells [10, 11]. But so far, no research has thoroughly clarified the pathogenesis of IDD and the ceRNA regulatory network of circRNAs remains to be explored. Therefore, this study will mine the microarray datasets in the GEO database to find differentially expressed RNAs and construct a circRNA-miRNA-mRNA ceRNA network to explore the pathogenesis of IDD and seek new targets with the aim of providing new options for treatment.

IDD is the pathological basis of a series of spinal degenerative diseases and the main cause of chronic low back and leg pain. Its pathological features are the degeneration of the cartilage endplate and the fibrosis of the nucleus pulposus, resulting in the decline of nutrients and cell viability in the intervertebral disc [12]. The pathological process of IDD involves multiple risk factors, including genetics, aging, autoimmune response, mechanical load, smoking, diabetes and so on, but the exact pathogenesis remains unclear. With the development of bioinformatics, more and more studies use this method to explore the pathogenesis of diseases, and provide reliable research directions and treatment suggestions for clinic. The ceRNA network constructed in this study included three down-regulated circRNAs(hsa_circ_0074817, hsa_circ_0002702, hsa_circ_0003600), three up-regulated miRNAs(hsa-miR-4741, hsa-miR-3158-5p, hsa-miR-508-5p) and 57 down-regulated mRNAs, of which six hub genes(IGF1, CHEK1, CCNB1, OIP5, BIRC5, AR) were identified by CytoHubba.

GO enrichment analysis showed that IDD involved multiple organisms, cells, molecules and was closely related to the components, binding modes and functions of cells. Biological processes showed that IDD was closely bound up with regulation of inflammatory response, positive regulation of cytokine production, endogenous cell promotion and other items. A number of current studies have shown that various genes can regulate the proliferation and apoptosis of intervertebral disc cells, or promote and inhibit the release of inflammatory factors to regulate the development of IDD [4]. The cell composition involved cell substrate junction. In intervertebral discs, extracellular matrix is the most important link, and the changes of its biochemical components directly lead to IDD [13]. Protein tyrosine kinase activity, ligand activated transcription factor activity and growth factor binding in molecular function were closely related to IDD. In this study, the hub gene IGF1 is a member of growth factors, which can affect the development of IDD through various ways.

KEGG pathway analysis showed that signalling pathways regulating pluripotency of stem cells and p53 signalling pathway were closely related to IDD, and both were involved in IGF1. IGF1 has been implicated in age-related diseases and is a key factor in anti-cellular aging, extending the lifespan of species. In vitro studies have reported that IGF1 can promote the synthesis of extracellular matrix. Further in vivo studies have found that the reduction of IGF1 bioavailability can reduce the senescence of intervertebral disc cells and the decomposition of extracellular matrix [14], and promote the synthesis of proteoglycan and type II collagen in intervertebral disc cells [15], which was consistent with the results of IDD development caused by the down-regulation of IGF1 in this study. As the largest avascular tissue in the whole body, the nutrient supply of intervertebral disc mainly comes from the infiltration of cartilage endplate, and IGF1 helps to increase the shuttle of nutrients. Kusuma found that human wharton's jelly mesenchymal stem cells induced by IGF1 can repair damaged articular cartilage by reducing inflammation and inhibiting MMP3 [16]. For the p53 signalling pathway, Kim found that senescent nucleus pulposus chondrocytes increased with age and promoted the development of IDD through the telomere-based p53-p21-pRB pathway [17]. At present, the role of IGF1 in the p53 signalling pathway is mainly focused on tumours. IGF1 regulates tumour development by affecting cell proliferation, apoptosis, and senescence through the p53 signalling pathway [18, 19]. The pathway was also involved in the hub genes CCNB1 and CHEK1. CHEK1 was also used in zhang's study as a hub gene and new biomarker for IDD [20], a serine/threonine-specific protein kinase that regulates the DNA damage response and cell cycle checkpoint response and influences tumour development through both of these [21]. The regulatory relationship between CHEK1 and p53 in apoptosis affects the development of various diseases [22, 23]. CCNB1 has also been found to be involved in the development of IDD by regulating the proliferation and apoptosis of nucleus pulposus cells [24], and it can also affect the cell cycle by silencing the p53 signalling pathway to induce tumour cells senescence and apoptosis [25].

The role of hsa-miR-508-5p upstream of IGF1, CHEK1 and CCNB1 in IDD remains to be explored, but it has been extensively studied in various tumour tissues and cardiovascular [26, 27]. Hsa_circ_0074817 can competitively sponge hsa-miR-508-5p to act as ceRNA, correcting the negative regulation of IGF1/CHEK1/CCNB1 by hsa-miR-508-5p. The upstream hsa-miR-4741 of BIRC5, OIP5 and AR has been reported as a core in ceRNA of IDD [28], but this regulation axis still needs to be experimentally verified for reliability and validity. This study suggests that hsa_circ_0003600-hsa-miR-4741-BIRC5/OIP5/AR regulatory axis may also be one of the potential mechanisms of IDD, but there is a lack of research support, as does the hsa_circ_0002702-hsa-miR-3158-5p-IGF1/AR regulatory axis, which are the direction and goal of our future research.

Inflammation runs through the development of IDD, and immune cells are the main cause of inflammation. This study showed that IDD was also inseparable from the infiltration of immune cells. The expression of T cells CD8 was significantly decreased in IDD, while the expression of neutrophils was significantly increased. Compared with healthy people, the percentage of CD8 + T cells in patients with lumbar degenerative diseases was significantly lower, and the percentage of CD4 + T cells was significantly increased, which may be related to the changes in lymphocyte subsets and increased apoptosis of CD8 + T cells after IDD immune response. Neutrophils make up 40%-70% of all white blood cells in humans and are an important part of the innate immune system, which can reach and concentrate on the degenerated intervertebral disc through blood vessels, and release a large number of inflammatory mediators to destroy the microenvironment of the intervertebral disc, thus promoting the development of IDD [29]. We also found that the expression level of certain hub genes also affected the infiltration of immune cells. CHEK1 inhibition can augment CD8 + T cells infiltration in vivo models of multiple immunocompetent small cell lung cancer [30]. CCNB1 and BIRC5 have been reported to be related to the immune infiltration of hepatocellular carcinoma, and their expressions are positively correlated with the infiltration levels of CD4 + T cells and dendritic cells, respectively [31, 32]. OIP5 is one of the ideal targets for tumour immunotherapy, and the amount of its expression determines the expression of apoptosis-related genes, which affects cancer progression [33]. In addition to inflammation, apoptotic Fas protein and its ligand FasL mediating apoptosis of Fas positive cells play an important role in intervertebral disc immune amnesty. Studies have shown that FasL expression is significantly reduced in degenerating discs, and that FasL and its potential immune privileging mechanism retard disc degeneration by preventing infiltration of host immune cells [34]. The hub genes IGF1, CHEK1, CCNB1, BIRC5 and OIP5 play a role in anti-senescence and apoptosis, whether they can reduce the senescence and apoptosis of FasL to improve the intervertebral disc microenvironment needs further study. Bone marrow mesenchymal stem cells have great potential and hope in the treatment of IDD because of their low immunogenicity and immune regulation. IGF1 can greatly enhance the bioactivity of human-derived mesenchymal stem cells and activate the PI3K/Akt signalling pathway to play a protective role against hypoxia and nutrient deficiency, thus providing guarantee for mesenchymal stem cells to treat IDD [35]. Thus, circRNAs, miRNAs and mRNAs not only directly affect IDD, but also by regulating immune cell infiltration.

There are some limitations in this study. First, the sample size of the datasets included in the study was small and there were some missing samples when performing immune analysis, which may have an impact on the accuracy and reliability of the results, and a larger sample size is needed to support it. Second, although some of the key genes of the present findings have been reported in previous studies, the same lack of experimental and clinical validation is a direction for further research.

In this study, we constructed and analysed the circRNA-miRNA-mRNA network by bioinformatics and found that the network not only mediates IDD by regulating cell senescence and apoptosis, but also affects IDD by influencing immune cell infiltration. These new findings will help us gain further insight into the pathogenesis of IDD and determine potential therapeutic targets.