Analysis of key genes and pathways associated with the pathogenesis of intervertebral disc degeneration

Low back pain (LBP) is increasingly recognized as a global public health problem associated with decreased quality of life and increased healthcare expenditure [1‐3]. It is estimated that 70–80% of the adult population suffer from at least one episode of LBP during their lifetime and 10% become chronically disabled [4]. With the changing of work and lifestyle, the incidence and prevalence of low back pain increase dramatically in the past few decades.

Intervertebral disc degeneration (IDD) is one of the most common sources of low back pain [5]. Being the largest avascular structure of the human body, the intervertebral disc is a complex structure consisting of the annulus fibrosus, nucleus pulposus, and cartilage endplate. The extracellular matrix (ECM) is the main component of the intervertebral disc which is responsible for maintaining both structure and function of the intervertebral disc. Intervertebral disc degeneration is characterized by the excessive degradation of ECM, leading to reduced hydration, loss of disc height, and decreased ability to absorb mechanical force [6].

Management of IDD includes both conservative treatment and surgery. Conservative management includes physiotherapy, medication, and epidural steroids injection. On the other hand, both fusion and non-fusion surgeries have demonstrated efficacy and safety in the management of IDD. The success rates of the Main and SPORT for surgically treated LDH patients and conservative-treated patients were 80% and 60%, respectively, at 1 year follow-up [7‐9]. Although both methods have achieved satisfactory clinical outcomes, neither conservative nor surgical treatment can completely resolve lumbar disc degeneration. Hence, it is necessary to elucidate the underlying mechanisms of IDD to find out a curative method. Previous studies showed that IDD is most likely to be multifactorial, including apoptosis [10, 11], inflammation [12, 13], aging [14, 15], and biomechanical loading [16, 17]. However, the genetic factors are regarded as the most significant contributor [18].

Nowadays, the gene chip technology and bioinformatics methods have been widely used to obtain the gene expression profile of the disease. This study uses bioinformatics methods to analyze the microarray of the nucleus pulposus (GSE23130) with the aim to identify differentially expressed genes (DEGs) and pathways related to the progression of IDD. We also investigated some hub genes involved in the progression of IDD based on the protein–protein interaction (PPI) network. For example, MMP3 has been reported as a key gene in maintaining homeostasis of the extracellular matrix, and in vivo study showed that gene therapy targeting MMP3 was an efficient way to delay intervertebral disc degeneration [19]. Hence, to screen out DEGs as potential target candidates is of significance for the prevention and treatment of IDD. This study may provide new insights into the pathogenesis of IDD and potential target candidates for new therapy.

Despite years of numerous clinical and experimental investigations, the underlying mechanisms of intervertebral disc degeneration remain unclear, which hinders the development of curative therapy. Genetic factors, mechanical factors, aging, inflammation, and other potential factors may cause IDD whereas genetic factors play a critical role based on published literatures. Several studies indicate that genetic factors are critical contributors to the onset and progression of IDD [25, 26]. For example, COL1A1 is a key gene encoding collagen I, and polymorphisms of the COL1A1 gene have been reported to increase the risk of IDD in different population studies [27, 28]. In the present study, we identify a total of 109 DEGs between degenerative samples and controls, including 79 upregulated and 30 downregulated DEGs.

In terms of GO enrichment analysis, we found that most of DEGs were mainly involved in skeletal system development, regulation of protein catabolic process, extracellular matrix (ECM) organization, collagen fibril organization, and extracellular structure organization. The extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network predominantly composed of collagens, proteoglycans, and many other glycoproteins. ECM is crucial for maintaining the structural and functional integrity of the intervertebral disc. Previous studies showed that even though many potential mechanisms induced intervertebral disc (IVD), they led to a final common result of excessive degradation of the extracellular matrix [29]. The imbalance between anabolism and catabolism of ECM is regulated by ECM-modifying enzymes such as matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors of metalloproteinases (TIMPs) [30‐32]. Lumican (LUM) is the most significantly upregulated gene in our analysis, which is one kind of keratan sulfate proteoglycan constituents of the ECM. Several studies showed that the abundance of lumican changed with the degeneration of the intervertebral disc [33, 34]. The study conducted by Vo et al. showed that ECM degradation increased by regulation of matrix metalloproteinases (MMPs) and ADAMTSs, leading to the development of IDD [35]. On the contrary, TIMP-1 and TIMP-2 mRNA and protein expression increase in degenerated IVD tissue, antagonizing the effect of MMPs [36]. Our bioinformatics analysis also showed that TIMP-1 increased in the IVD samples than controls.

Regarding the KEGG pathway of our analysis, we found that most DEGs were primarily enriched in the PI3K-Akt signaling pathway. The PI3K-Akt signal pathway showed protective effects on the human nucleus pulposus under different pathological conditions. Activation of the PI3K-Akt pathway protects against IDD by the increase of ECM content, prevention of cell apoptosis, and induction or prevention of cell autophagy. Studies conformed that the activation of the PI3K-Akt pathway increased the SOX9 expression and activity and consequently led to the increase of the aggrecan expression in NP cells [37]. A study revealed that 17β-estradiol (E2) prevented the degradation of ECM by the activation of PI3K-Akt-FOXO3, which reduced the expression of MMP-3 and increased the expression of collagen II and aggrecan expression [38]. Many recent studies also showed that resveratrol suppressed IL-1β-mediated NP cell apoptosis through activating the PI3K-Akt pathway [39‐41]. On the contrary, as the only known lipid phosphatase, tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) can counteract the protective effect of the PI3K-Akt pathway. Xi et al. showed that PTEN promoted intervertebral disc degeneration by negatively influence PI3K-Akt [42]. Hence, gene therapy targeting PTEN may play an important role in treating IDD.

We further constructed a PPI network for better understanding of the interaction between DEGs. The most significant module was extracted from the PPI network using the MCODE plugin. Furthermore, the top 10 hub genes—FN1, COL1A2, SPARC, COL3A1, CTGF, LUM, TIMP1, THBS2, COL5A2, and TGFBI—were identified from this network. To be mentioned, all hub genes were also enriched in the most significant module. TGFBI is the seed DEG of the module. TGFBI is a protein secreted by many types of cells. It binds to collagen, forms part of the extracellular matrix (ECM), and interacts with integrins on cell surfaces. The study showed that TGF-β increased the expression of COL1A1, ACAN, and SOX9 genes by mediating communication between nucleus pulposus cells and mesenchymal stem cells [43]. Activation of TGF-β signaling has a protective effect on the intervertebral disc via inhibition of ECM degradation and increase of ECM synthesis, promotion of cell proliferation and inhibition of cell death, and alleviation of inflammatory response. However, excessive activation of TGF-β signaling may contribute to IVD degeneration [44]. SPARC is a matricellular glycoprotein involved in interactions between cells and matrices. Gruber showed that the deletion of the SPARC gene accelerated disc degeneration in the aging mouse [45]. Millecamps et al. demonstrated that inactivation of the SPARC gene led to the early onset of both disc degeneration and behavioral indices of LBP in mice [46]. Tajerian et al. showed that the underlying mechanism of the silence of the SPARC gene during aging may be attributed to DNA methylation [47]. A recent in vivo experimental study showed that stable expression of CTGF and TIMP1 genes by co-transfection adeno-associated virus 2 increased synthesis of aggrecan and type II collagen in the degenerated intervertebral disc, which served as a potential target gene for disc regeneration [48]. Thrombospondin proteins (THBSs) are a class of glycoproteins that function in maintaining homeostasis of ECM by regulating the level of matrix metalloproteinase-2 (MMP-2) and MMP-9 [49, 50]. A Japanese population-based genetic and functional data indicated that THBS2 played an important role in the pathogenesis of LDH by acting as a modulator of MMP-2 and MMP-9 endocytosis [51].

In summary, the present study provides a comprehensive analysis about the pathogenesis of IDD and offers potential target genes for the early diagnosis and treatment of intervertebral disc disease. The PI3K-Akt signal pathway and related hub genes may play important roles in the progression of IDD that needs deeper investigation. Nevertheless, further experiments are required to validate their effects and mechanisms in IDD.