Despite years of numerous clinical and experimental investigation, the underlying mechanisms of intervertebral disc degeneration remains 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 COL1A1 gene has 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. 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 structural and functional integrity of intervertebral disc. Previous studies showed that even though many potential mechanisms induced IVD, they led to a final common result of excessive degradation of the extracellular matrix. 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 up-regulated 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 intervertebral disc[33,34]. Study conducted by Vo NV et al. showed that ECM degradation increased by regulation of matrix metalloproteinases (MMPs), and ADAMTSs , leading to the development of IDD. On the contrary, TIMP-1 and TIMP-2 mRNA and protein expression increases in degenerated IVD tissue, antagonizing the effect of MMPs. Our bioinformatic analysis also showed that TIMP-1 increased in the IVD samples than controls.
Regarding KEGG pathway of our analysis, we found that most of DEGs were primarily enriched in PI3K-Akt signaling pathway. PI3K-Akt signal pathway showed protective effects on human nucleus pulposus under different pathological conditions. Activation of PI3K-Akt pathway protects against IDD by increase of ECM content, prevention of cell apoptosis and induction or prevention of cell autophagy. Studies conformed that activation of PI3K-Akt pathway increased SOX9 expression and activity, and consequently led to increase of aggrecan expression in NP cells. A study revealed that 17β-estradiol (E2) prevented the degradation of ECM by activation PI3K-Akt-FOXO3, which reduced the expression of MMP-3 and increased the expression of collagen II and aggrecan expression. 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 PI3K-Akt pathway. Xi Y et al. Showed that PTEN promoted intervertebral disc degeneration by negatively influence PI3K-Akt. 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 MCODE plugin. Furthermore, 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 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. Study showed that TGF-β increased expression of COL1A1, ACAN, and SOX9 genes by mediating communication between nucleus pulposus cells and mesenchymal stem cells. Activation of TGF-β signaling has a protective effect on 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-βsignaling may contribute to IVD degeneration. SPARC is a matricellular glycoprotein involved in interactions between cells and matrix. Gruber HE et al. showed that deletion of the SPARC gene accelerated disc degeneration in the aging mouse. Millecamps et al. demonstrated that inactivation of the SPARC gene led to early onset of both disc degeneration and behavioral indices of LBP in mice. Tajerian M et al. Showed that the underlying mechanism of silence of SPARC gene during aging may attributed to DNA methylation. 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 degenerated intervertebral disc, which served as potential target gene for disc regeneration. Thrombospondin proteins (THBSs) are a class of glycoproteins that functions in maintaining homeostasis of ECM by regulating 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 modulator of MMP-2 and MMP-9 endocytosis.