IVDD is an important cause of cervical and lumbar disc herniation and low back pain(Meng, Huang, 2021). During the progression of the disease, the intervertebral disc fibrous annulus (AF) mislays its original structural level and toughness and develops reticular degeneration and hyalinisation, while the water content of the nucleus pulposus (NP) decreases, eventually manifesting as a loss of normal elasticity and tone of the disc(Roberts et al., 2006). But how does CS play a role in IVDD? It is generally accepted that senescent cells in the IVDD contribute to disc degeneration through the secretion of SASP, which promotes the senescence of neighboring cells, mediates the secretion of matrix proteases, promotes immune cell infiltration, and enhances the inflammatory microenvironment, ultimately leading to an imbalance between ECM production and catabolism(Patil et al., 2018). Unfortunately, the exact composition ofSIPSsecreted by senescent cells in the intervertebral disc and the exact mechanism of action are not yet known. It is also known that DNA damage, oxidative stress, inflammation, and mechanical stress may contribute to CS in intervertebral disc cells, but the exact mechanism of action remains unclear. More scholars are now targeting senescent cells to treat degenerative diseases and point to this as a promising therapeutic strategy(van Deursen, 2019). For example, a study by Guo et al. showed that resveratrol, a SIRT1 activator, inhibited apoptosis, matrix protease (MMP13 and ADAMTS5) activity and p16INK4a and p21Cip1 expression in human NP cells in degenerating discs(Guo et al., 2017). Our study aims to apply CSRG to predict the prognosis and subtype classification of IVDD in order to guide the prevention and treatment of IVDD.
In this study, we first obtained 53 differentially expressed CSRGs out of 278 CSRGs by differential analysis of IVDD patients and non-IVDD patients. We then obtained four hub genes (DUSP3, MAPKAPK5, SP1, and VEGFA) by combining the MECOD algorithm, LASSO analysis, RF algorithm, and SVM-RFE algorithm. To predict the prevalence of IVDD patients, we built a nomogram model based on these four hub genes. DUSP3 is an atypical protein tyrosine phosphatase that dephosphorylates ERK, JNK, p38, tyrosine phosphorylated insulin receptors, epidermal growth factor receptors, and keratin-forming cell growth factor receptors (IR, PDGFR, EGFR, and KGFR, respectively), platelet-derived growth factor receptors, receptor tyrosine kinases, and serine phosphorylated casein(Amand et al., 2014, Pavic et al., 2015). In addition, DUSP3 plays an important role in DNA damage response, cell cycle regulation, angiogenesis, MAPK signaling, and platelet activation, and therefore promotes or inhibits a variety of tumorigenic diseases. Wang et al. showed that the mRNA and protein levels of VHR and its activity on MAPK were lower in non-small cell lung cancer tissues than in normal tissues, and thus epigenetic suppression of VHR may contribute to the initiation of lung carcinogenesis(Wang et al., 2011). In another study, VHR levels were found to be higher in epithelial cell lines of different grades of squamous and adenocarcinoma from cervical tissue than in primary cells and normal tissue, and were mainly enriched in the nucleus(Henkens et al., 2008)(34). MK5 is a serine/threonine protein kinase, also known as p38-activated protein kinase or PRAK, because it can be phosphorylated and activated by p38 MAP kinase(New et al., 1998). MK5 phosphorylates FOXO3, ERK3/MAPK6, ERK4/MAPK4, HSP27/HSPB1, p53/TP53 and RHEB to play a dual role in inhibiting or promoting tumour progression(Perander et al., 2016). Sp1, the first transcription factor identified in the Sp/KLF family, regulates cell proliferation, differentiation, apoptosis, and angiogenesis by binding to GC-rich structural domains in the regulatory regions of genes encoding components of the p38, MAPK, PI3K/Akt, and JAK/STAT signalling pathways(Ivanenko et al., 2022). Recently, it has been discovered that SP1 is intimately linked to a number of neoplastic conditions, such as breast, pancreatic, lung, and thyroid cancer, as well as neurological conditions including Parkinson's disease and Huntington's(Ivanenko, Prassolov, 2022). VEGFA was originally defined as an endothelial growth factor as well as a vascular permeability regulator that can be produced by most cells in the body and is also known as vascular permeability factor(Safe et al., 2018). VEGFA can induce various VEGFR2 downstream signalling pathways including the phospholipase Cc-extracellular regulated kinase pathway, Src kinase, adherens plaque kinase, PI3K-PKB) Akt pathway and monomeric G protein Rho family to promote angiogenesis(Claesson-Welsh and Welsh, 2013). Zhan et al. found that VEGFA was involved in the process of disc degeneration in rabbits due to sustained compression and proposed that modulating VEGFA expression could improve the lesioning of vascular buds in the disc and enhance nutrient supply, ultimately preventing and treating IVDD effectively(Zhan et al., 2020). However, the role and mechanism of these four hub genes in disc degeneration is not yet clear, and we hope that our study will provide direction for future studies of these four hub genes in disc degeneration.
It is now generally accepted that the development of IVDD is closely linked to immunity and inflammation. Under normal physiological conditions, the NP is strongly isolated from the circulatory and immune system by the blood NP barrier (BNP), consisting of the AF in the outer layer of the NP, the CEP overlying the upper and lower parts and the secretory inhibitor of angiogenesis, which also makes the intervertebral disc an immune privileged organ(Sun et al., 2020). However, when AF or CEP fissures or ruptures during disc degeneration, a variety of immune cells, including T cells (CD4+, CD8+), B cells, macrophages, neutrophils, and mast cells, will migrate into the disc(Risbud and Shapiro, 2014). The influx of immune cells into the disc will cause the expression of cytokines and chemokines to rise even more, encouraging inflammation-driven catabolism and ultimately speeding up the degradation of the ECM(Phillips et al., 2015, Risbud and Shapiro, 2014). In addition, several cytokines, including TNF-α, IL-1β, IL-6, IL-10, IL-4, TGF-β, and IFN-γ, also play an important role in intervertebral disc degeneration(43, 44). For example, a study by Wang et al. found that treatment with IL-1β or TNF-α increased the secretion of CCL3 from senescent NP cells and in turn increased the level of macrophage infiltration(Phillips, Cullen, 2015, Risbud and Shapiro, 2014). In our study, two hub gene clusters (hub gene clusters A and B) were identified by consistent clustering based on four hub genes. We then analysed cluster A and found higher levels of infiltration of immune cells such as activated dendritic cells, CD56dim natural killer cells, immature dendritic cells, macrophages, monocytes, type 1 T helper cells, and plasmacytoid dendritic cells, suggesting that hub gene cluster A is associated with the development of IVDD. We then confirmed the reliability of these results based on 297 DEGs between two hub gene clusters in the gene model. To quantify the hub gene pattern, we used the PCA algorithm to calculate the hub score for each sample. We found that hub gene cluster A or gene cluster A had a higher hub score than hub gene cluster B or gene cluster B. Finally, we used qPCR analysis and western blot to verify the expression levels of mRNA and protein in normal and IVDD cells of 4 hub CSRGs.