The pathological mechanisms of circRNAs in mediating intervertebral disc degeneration

Lower back pain (LBP) is a worldwide health problem associated with significant economic and social burden. Intervertebral disc degeneration (IVDD) is a leading cause of LBP. Several studies show that the death of nucleus pulposus cells (NPCs), abnormal metabolism of the extracellular matrix (ECM), and inflammatory response are the key mechanisms behind the pathogenesis of IVDD. Circular RNAs (circRNAs) are key regulators of gene expression and play a significant role in regulating NPCs death, ECM homeostasis, and inflammatory response by acting as microRNAs (miRNAs) sponges in IVDD. However, the regulatory role of circRNAs in mediating IVDD remains unknown. This review comprehensively describes the normal anatomic structure and function of IVD, the pathogenesis of IVDD, the characteristics, synthesis, mechanisms, and function of circRNAs. Moreover, we highlighted the 23 circRNAs that mediate ECM metabolism, 16 circRNAs that mediate NPCs apoptosis, circ_0004354 and circ_0040039 that mediate NPCs pyroptosis, and 5 circRNAs that mediate inflammatory response in IVDD. In addition, this review presents suggestions for future studies, such as the need for further investigation on ferroptosis-related circRNAs in IVDD. This review could provide novel insights into the pathogenesis and treatment of IVDD.


Introduction
Low back pain (LBP) is a common worldwide health problem in adults, associated with a huge economic and social burden to patients, families, and the society [1].A previous systematic analysis conducted in 2019 on the global burden of 369 diseases in 204 countries revealed that LBP was the fourth cause of disability among people aged 25-74 years [2].The Lancet series call for worldwide recognition of LBP.Furthermore, the prevention, diagnosis, treatment, and rehabilitation of patients with LBP is associated with various challenges [2][3][4][5][6].Intervertebral disc degeneration (IVDD) is reported to be one of the main causes of LBP, with about 40% of LBP cases attributed to intervertebral disc degeneration (IVDD).
Intervertebral disc degeneration can be caused by various internal factors such as genetics and aging and external factors such as infections, trauma, and smoking.Genetic factors account for 34-61% of IVDD cases [4,[7][8][9].The nucleus pulposus (NP) tissue is located at the center of the intervertebral disc (IVD) and is mainly composed of nucleus pulposus cells (NPCs) and extracellular matrix (ECM).The NP is a gel-like substance composed of water, proteoglycans, elastin fibers, and proteins.The NP distributes hydraulic pressure throughout the intervertebral disc.Degeneration of the intervertebral disc usually starts from the nucleus pulposus.The NP comprises nucleus pulposus cells (NPCs) and an extracellular matrix (ECM).Furthermore, the physiological function of the nucleus pulposus depends on the homeostasis of its microenvironment and inflammatory response.
The NP lacks blood supply and self-repair ability.Disc degeneration could occur due to inflammation, ischemia, hypoxia, and acidic microenvironment [10].The current management of IVDD aims to alleviate symptoms but does not reverse the degeneration.Moreover, surgery impairs the integrity of intervertebral discs, leading to accelerated degeneration, recurrence, and adjacent segment degeneration [11][12][13].Gene therapy targeting the nucleus pulposus could block or reverse the pathological process of IVDD, thus offering a promising effective therapeutic strategy for IVDD [6,14].
Circular RNAs (circRNAs) are a major type of regulatory non-coding RNA, which can mediate the occurrence and development of IVDD by sponging microRNAs (miRNAs) and are involved in posttranscriptional gene regulation.circRNAs could be exploited as novel diagnostic and prognostic biomarkers.This review provides an update on the regulatory function of circRNAs in the degeneration of the nucleus pulposus.This review aims to provide a theoretical basis for understanding the potential therapeutic targets for developing novel therapies for IVDD.

The normal anatomic structure and function of IVD
The human spine comprises 23 vertebrae, including 6 cervical vertebrae, 12 thoracic vertebrae, and 5 lumbar vertebrae, accounting for about a quarter of the total height of the spine [15].Each intervertebral disc can bear complex mechanical loads while maintaining spinal flexibility, including axial bending, compression, and rotation [16].The intervertebral disc is a fibrocartilage located between two adjacent vertebrae, and is an important functional unit of the spine.It consists of a central gelatinous core called the nucleus pulposus, annulus fibrosus (AF) surrounding the nucleus pulposus, and the cartilage endplate that connects adjacent vertebrae [17].The intervertebral disc is an avascular organ whereby blood vessels end at the cartilage endplate and do not cross through the nucleus pulposus or the inner layer of annulus fibrosus.Therefore, the nucleus pulposus lacks blood supply and exists in a hypoxic-ischemic microenvironment.However, oxygen and nutrients enter into the nucleus pulposus and annulus fibrosus through the cartilage endplate [18].The cartilage endplate contains channels responsible for discharging metabolic wastes from the intervertebral disc [19,20].
The extracellular matrix (ECM) is a non-cellular, complex, and highly dynamic structure, that regulates cell function and promotes cell communication.The ECM plays an important role in maintaining the integrity and biomechanical function of intervertebral discs.In addition, the ECM can selectively bind to growth factors related to tissue morphogenesis, homeostasis, and repair [21].The ECM of the nucleus pulposus is mainly composed of aggregate proteoglycans (ACAN) and collagen type II (COL2) in a ratio of 27:1.with COL2 accounting for about 20% of the dry weight of the nucleus pulposus.On the other hand, ACAN is the main proteoglycan of the nucleus pulposus, accounting for about 50% of the wet weight of the nucleus pulposus [10].The proteoglycans are rich in chondroitin sulfate and are aggregated with hyaluronic acid, which helps maintain osmolar pressure in the nucleus polposus and helps the disc act as a shock absorber [10,22,23].
Previous studies reveal that ACAN is important in regulating the growth of nerves and blood vessels in the intervertebral disc [22][23][24].For example, Stefanakis et al. [24] showed that IVD degradation and depletion of ACAN were associated with an influx of blood vessels and nerves.COL2 forms the collagen fiber network, which holds proteoglycans and provides the intervertebral disc with tensile strength [25].Factors hindering the diffusion of oxygen and nutrients into the vertebral disc or discharge of metabolic wastes could lead to the reduction of oxygen tension in the intervertebral disc and anaerobic metabolism, thus leading to increased production of lactic acid and decreased PH.Consequently, this could affect the metabolic and biosynthetic function of NPCs thus impairing ECM synthesis and decomposition.

The pathogenesis of IVDD
The pathological process of IVDD is divided into three stages (Fig. 1): ① Stage 1 involves the secretion of proinflammatory mediators by nucleus pulposus cells (NPCs) due to various factors such as heredity, aging, infections, trauma, and smoking, and the high content of ECM and the low content of matrix metalloproteinases (MMPs), chemokines in intervertebral discs; ② The released proinflammatory factors cause apoptosis and pyroptosis of NPCs and further release of inflammatory factors; NPCs also secrete chemokines, which cause the activation and infiltration of CD4+and CD8+T cells, M1 macrophages, mast cells, neutrophils, and other immune cells, which also release more cytokines and chemokines.These cytokines and chemokines recruit more immune cells into the intervertebral disc, which secrete proinflammatory cytokines leading to an inflammatory cascade.In this stage, there is still a high ECM in the intervertebral discs.③The proinflammatory factors and the inflammatory cascade promote the expression of matrix metalloproteinases (MMPs), leading to ECM degradation、reduced intervertebral discs height, and annulus fissure, thus causing IVDD [4][5][6]26].Progression of IVDD is associated with decreased water content of the nucleus pulposus tissue, decreased diffusion of oxygen and nutrients, and decreased discharge of metabolic wastes from the intervertebral disc.These factors worsen the hypoxic environment in the intervertebral disc and lead to anaerobic metabolism, thus increasing lactic acid production and accumulation and decreasing the pH value of the microenvironment, further promoting NPCs death and ECM degradation [10,27].The altered homeostasis, ECM degradation, and inflammation of intervertebral disc lead to changes in the morphology (the height of the intervertebral disc decreases) [28], growth of microvessels and nerve fibers into the intervertebral disc [4,23,24,26,29]), decrease water content in the intervertebral disc [30]), lead to loss of flexibility and elasticity [31], and cause biochemical alterations [4][5][6]26] of the intervertebral disc.Therefore, the key pathogenic mechanisms behind IVDD include the increased apoptosis of NPCs in the microenvironment of intervertebral discs [32], the enhanced inflammatory response [4][5][6]26], and increased ECM degradation [10,33].
Seven types of circRNAs have been identified based on their splice positions in the genome [42], including ecircRNA (from exonic regions), ciRNA (from intronic regions), eiciRNA (from both exonic and intronic regions), read through circRNA (rt circRNA) formed by cyclization of two exons of two different genes, tricRNA formed by pre-tRNA intron splicing [51], fusion gene derived f-circRNA [52], and mecciRNA encoded by the mitochondrial gene [53].Among them, ecircRNA is the most common type, which is spliced from at least one exon of a single gene.ecircRNAs are usually located in the cytoplasm, where they function as miRNA sponges [42].
Among the 12 circRNAs that promote ECM anabolism, circ-4099 was shown to be significantly up-regulated in IVDD, while the other 11 circRNAs were significantly down-regulated in IVDD.
Y. Li et al.
Pyroptosis is a type of regulated inflammatory cell death executed by Gasdermin family proteins, which form pores on the cell membrane.The activation of inflammatory CASPs promotes the secretion of IL-1β and IL-18 in pyroptosis [91].Zhang et al. [92] showed significantly decreased expression of miR-410 in needle puncture-induced IVDD models.miR-410 is a key negative regulator of NPCs pyroptosis.Xu et al. [93] reported that miR-141 was significantly overexpressed in IVDD, which induced NPCs pyroptosis, ECM catabolism, and inflammation by increasing ROS production and stimulating the TXNIP-NLRP3 signaling axis.Interestingly, under TNF-α induction, circ_0004354 and circ_0040039 promoted the expression of cysteine aspartate proteinase 3 (CASP3) and GSDME to varying degrees, which participated in the regulation of NPCs pyroptosis and promoted the secretion of IL-1β [63] (Table 1 and Fig. 2).
Ferroptosis, an iron-dependent non-apoptotic and non-pyroptotic cell death, accompanied by lipid peroxidation and increased reactive oxygen species (ROS), was first reported by Dixon et al. [94].Ferroptosis is related to the pathogenesis of IVDD.In addition, previous studies showed that inhibiting ferroptosis could be exploited as an effective strategy to delay IVDD [95][96][97].GPX4 is an important marker of ferroptosis.A previous study showed decreased levels of GPX4 and ferritin heavy chain in a rat model of IVDD [95].Sheng et al. [98] showed that the IL-6-miR-10a-5p-IL-6R signaling axis plays an essential role in regulating IVDD.Il-6 inhibits the expression of IL-6R by inhibiting miR-10A-5p, thereby inhibiting IL-6-induced ferroptosis.Previous studies showed that circRNAs mediate ferroptosis by regulating key proteins.In addition, circRNAs play an important role in various diseases, including cancer and traumatic brain injury [98,99].However, circRNAs-mediated ferroptosis in IVDD has rarely been reported and needs further investigation.

Inflammatory reaction
The inflammatory reaction is caused by overexpression of inflammatory cytokines.Intervertebral disc degeneration is characterized by elevated levels of inflammatory cytokines, such as TNF-α, IL-1α, IL-1β, IL-6, IL-17, IL-18, and IFN-γ, and an inflammatory cascade, which promotes the production of matrix metalloproteinase and other catabolic factors that induce ECM degradation and enhance cell death.Impairment in the normal physiological function of the intervertebral disc may cause IVDD, painful nerve fibers, and growth of microvascular vessels into the intervertebral disc, leading to low back pain [4][5][6]26,93,100].Furthermore, available evidence reveals that dysregulation of circRNAs expression is associated with the production of inflammatory cytokines during IVDD.Five circRNAs have been reported to regulate the inflammatory response in IVDD, including four proinflammatory circRNAs (circ_0004354 [63], circ_0040039 [63], circ-FAM169A [66], and circRNA_0000253 [67]) and one anti-inflammatory circRNAs: circ-4099 [83] (Table 1 and Fig. 2).Cheng et al. showed that circRNA VMA21 promotes XIAP expression by binding miRNA-200C [77].Increasing evidence reveals that XIAP can regulate the inflammatory response, while XIAP inhibition can promote the excessive secretion of TNF-α and IL-1β [101,102].However, further studies are needed to investigate whether circRNA VMA21 regulates XIAP or mediates inflammation in IVDD.Fig. 2. Different circRNAs act as protector or inhibitor in IVDD by regulating ECM metabolism, apoptosis, pyroptosis, and inflammatory response.In the upper half of the ellipse, 10 circRNAs promote ECM degradation; 9 circRNAs promote cells apoptosis; 2 circRNAs promote cells pyroptosis; 4 circRNAs promote inflammatory response; thus, these IVDD-related circRNAs were regarded as IVDD-inhibitor.In the lower half of the ellipse, 12 circRNAs promote ECM synthesis; 7 circRNAs inhibit cells apoptosis; 2 circRNAs inhibit inflammatory response; thus, these IVDD-related circRNAs were regarded as IVDD-protector.

Conclusion
Intervertebral disc degeneration is the main cause of low back pain, which affects the physical and mental health of patients.IVDD begins in the nucleus pulposus and is associated with various pathological mechanisms of NPCs, including apoptosis, pyroptosis, ferroptosis, inflammation, and ECM degradation.Recent studies showed that circRNAs regulate the expression of key proteins, including transcription factors related to IVDD, by acting as miRNA sponges, mediating NPCs death, inflammation, and ECM-related signaling pathways.In addition, circRNAs are involved in the pathological process of IVDD.
However, our understanding of circRNAs is still limited, and several questions remain unanswered.First, several circRNAs have been shown to play crucial roles in IVDD.However, studies have not revealed the key circRNAs involved in IVDD.Second, it is not clear whether different key circRNAs play different roles at different pathological stages of IVDD.Third, the upstream mediators involved in the dysregulation of the expression of most circRNAs in NPCs and their association with various risk factors of IVDD, such as genetics, aging, smoking, and trauma, remain largely unknown.In addition, it is not clear what factors affect the expression and function of circRNAs.Fourth, only a few studies report on the circRNAs that are dysregulated in other cell types associated with IVDD, such as cartilage endplate chondrocytes and annulus fibrosus cells.Fifth, it has not been reported whether circRNAs mediate IVDD progression through coding proteins, regulatory protein translation, parental gene transcription, or interaction with RNA-binding proteins.Therefore, further studies are needed to clarify this.Sixth, only a few studies report on the circrNA-circrNA interaction.Seventh, the use of serum circRNAs as diagnostic IVDD markers has not been reported.In the future, large-scale screening and validation of serum circRNAs related to diagnosis and prognosis should be carried out.Eighth, how circRNAs could be applied in the clinical setting to treat IVDD disease remains a huge challenge.Patients with early-to mid-stage IVDD may not present with severe symptoms.Therefore, there is a need to identify IVDD in the early stages to prevent IVDD progression and implement early interventions for the treatment of IVDD, thus improving patients' quality of life.

Fig. 1 .
Fig. 1.The pathological process of IVDD.(A) Secretion of proinflammatory factor by nucleus pulposus cells; (B) Apoptosis of nucleus pulposus cells and inflammatory response; (C) ECM degradation caused by MMPs.