Intervertebral disc degeneration (IDD) is characterized by increased extracellular matrix breakdown and abnormal matrix synthesis leading to reduced hydration, loss of disc height, and decreased ability to absorb load (1,2). It is considered to be the primary source of chronic lower back pain and spine-associated disease, which leads to major economic and social burdens that affect millions of individuals globally (3). The major clinical manifestations of IDD are disc herniation, vertebral instability and spinal stenosis. The ability to treat IDD effectively is hindered by an incomplete understanding of the biological processes that control intervertebral disc development, function and disease. At present, IDD continues to be treated with symptomatic interventions, which do not lead to substantially improved outcomes, as no disease-modifying drugs are currently available (4). Consequently, the clinical management of IDD pathologies remains severely limited, with no options at present for early intervention or predictive patient screening. Therefore, an improved understanding of the pathophysiology and molecular mechanisms underlying IDD is essential for diagnosis and the development of novel therapeutic approaches.

Although the etiology of IDD is likely to be multifactorial, genetic factors are considered to be the greatest contributors (5). Recently, the molecular basis of degenerative disc disease has received increased attention in research, which has substantially improved the understanding of the biology underlying this process. Studies that employed classic experimental approaches to investigate the molecular changes associated with the pathophysiology of IDD have established criteria to define degenerative intervertebral discs (5,6). While helpful, these criteria involve relatively few factors. In recent years, there has been an increase in the use of transcriptomic approaches to identify the large spectrum of factors that exhibit altered expression during IDD. For instance, Chen et al (7) identified mitogen-activated protein kinase kinase 6 and Rho-related BTB domain-containing 2 as two specific therapeutic molecular targets in the treatment of IDD. Periostin was proven to be upregulated in the progression of human IDD (8). Furthermore, high-throughput screening of human patient samples may identify potential biomarkers of IDD, leading to more precise diagnostic criteria, classification of disease progression and prognosis (9).

The intervertebral disc is composed of specialized connective tissue structures that link adjacent vertebral bodies along the spine and confer flexibility and mechanical stability to the body trunk during axial compression. There are three morphologically distinct regions in the intervertebral disc; the nucleus pulposus (NP), annulus fibrosis (AF) and cartilaginous endplates (10). Previous microarray analysis of mRNA isolated from AF cells identified differential expression of insulin-like growth factor binding protein 3 and interferon-induced protein with tetratricopeptide repeats 3 in the AF of IDD samples when compared with the control samples (11). However, the results obtained were limited as the study did not contain samples from NP, which is an important region of the human intervertebral disc. Therefore, the reanalysis of the gene expression profile by applying bioinformatics methods remains necessary to identify differentially expressed genes (DEGs) in IDD and further elucidate the potential pathogenesis mechanisms of the disease.

The present study aimed to identify the DEGs and further analyze their functions and pathways associated with the progression of IDD by utilizing a bioinformatics method to analyze microarray expression profiles from the NP and AF, and to obtain additional insights regarding the mechanisms of IDD.

Despite years of investigation, the pathogenesis underlying IDD remains poorly understood and continues to require further investigation. The emergence of bioinformatics methods has accelerated the progress of research on the mechanisms of human disease. The present study identified 93 and 114 DEGs in the NP and AF respectively, through the comparative analysis of the transcriptome between degenerative intervertebral disc samples and controls. The analysis identified 35 common DEGs in the two regions, and protein-protein interaction network modules demonstrated that the inflammatory cytokine interferon signaling may serve an important role in human IDD. In addition, a total of 6 associated miRNAs (miR-96, miR-182, miR-31, miR-526B, miR-188 and miR-19) were identified, which targeted these common DEGs.

Alterations in the production of extracellular matrix and inflammatory cytokines by intervertebral discs have an important role in the pathogenesis of IDD (5). In the present study, functional enrichment analysis indicated a number of DEGs involved in biological processes, including cell adhesion, biological adhesion and extracellular matrix organization, in the two regions of the intervertebral disc. Pathway enrichment analysis demonstrated that focal adhesion and the p53 signaling pathway were disrupted in NP and AF. In addition, 3 common DEGs in the regulatory network were enriched in extracellular matrix organization and 4 common DEGs in the regulatory network were enriched in the focal adhesion signaling pathway.

The extracellular matrix is a component of all mammalian tissues, and is a network that consists predominantly of the fibrous proteins collagen, elastin and fibronectin. In addition to a structural function, the extracellular matrix exhibits a number of other roles; as a major component of the cellular microenvironment, it affects various cell behaviors, which include proliferation, adhesion and migration, and also regulates cell differentiation and death (21). Extracellular matrix composition is particularly heterogeneous and dynamic, and abnormal extracellular matrix dynamics may lead to dysregulated cell proliferation, cell death failure and loss of cell differentiation, which subsequently results in congenital defects and pathological processes such as tissue fibrosis and cancer (22). It has been reported that during IDD, the ability of intervertebral disc cells to produce extracellular matrix reduces, however, the production of degradative enzymes does not change; this phenomenon is hypothesized to accelerate the degeneration by degrading the extracellular matrix of the disc, ultimately resulting in the macroscopic changes of the intervertebral disc (6).

Collagen, which gives tissues the ability to recover following stretching, is the most abundant fibrous protein within the extracellular matrix. It has been identified that the integrin-binding sialoprotein (IBSP) interacts with collagen and appears to modulate cell-matrix interactions (23). Cell-matrix adhesions have essential roles in a number of important biological processes, including cell motility, proliferation and differentiation, and the regulation of gene expression and cell survival; at contact points between the cell and extracellular matrix, specialized structures termed focal adhesions are formed (24).

The RAP1A gene encodes a member of the Ras family of small GTPases. Alterations in the conformation and activity of the protein encoded by RAP1A occur depending on whether GTP or GDP is bound to the protein, which are involved in regulating signaling pathways that affect cell proliferation and adhesion (25,26). Therefore, the dysregulation of collagen type VI α2 (COL6A2), IBSP and RAP1A in human intervertebral discs may induce de-adhesion, characterized by disruption of extracellular matrix organization and focal adhesions, which accelerates the degeneration of intervertebral discs.

Since the discovery of interferons, they have been investigated widely in a large number of studies, and considerable progress has been made in describing the nature of the cytokines themselves (27). Originally, interferons were known for their antiviral properties, however, interferons are currently better known for their distinct cellular functions, which include inhibition of proliferation and angiogenesis, induction of differentiation and regulation of the immune system (28). Previous research has demonstrated that IFIT3 may lead to AF cell growth arrest via its antiproliferative activity, which negatively regulates the cell cycle and directly or indirectly induces cell apoptosis (11,29,30). This is similar to the results of the present study. However, the difference and innovation of the present study primarily lies in the different groups included in the differential analysis. The present study included two sets of differential analysis, between the NP and control groups and the AF and control groups, respectively, while in the study by Kazezian et al (11), the differential analysis was only conducted between the NP and AF groups, and so the DEGs were not exactly the same. According to the results of protein-protein interaction network modules presented in the present study, three interferon-induced genes (IFIT1, IFIT2 and IFIT3) were enriched in the degenerative human discs. Therefore, based on the above information, the upregulated IFITs may negatively regulate the cell cycle, and thus reduce the disc cell number, subsequently accelerating degeneration.

miRNAs are considered to serve a crucial role in gene expression, which affects numerous biological processes, including cell differentiation, proliferation, metabolism, apoptosis and tumorigenesis, and have become therapeutic targets for diseases such as IDD (31–33). To investigate the potential molecular targets, miRNAs interacting with the common DEGs were retrieved in the present study and a regulatory network was also constructed. Aberrant expression of miR-96 or miR-182 has been reported in a number of human diseases, including pulmonary arterial hypertension and cancer (34–37). The results of the present study, and of previous studies, have demonstrated that forkhead transcription factor F2 (FOXF2) may be regulated by miR-96 or miR-182 (38,39). FOX is a super family of transcriptional regulators that exhibit numerous functions in human diseases (38–40). The FOXF subfamily consists of two members, FOXF1 and FOXF2. A previous study indicated that FOXF2 promoted extracellular matrix production, and in FOXF2 mutant animals, the extracellular matrix, particularly collagens, was severely reduced, which causes tissue disintegration (41). In addition, FOXF2 may act as a mesenchymal factor that controls cell proliferation and survival (42). Therefore, we hypothesized that FOXF2, miR-96 and miR-182 are worthy of further investigation to determine their specific roles in IDD.

In conclusion, the present study provides integrated network insight into the pathogenesis of IDD and offers potential therapeutic targets for controlling the disease. The dysregulation of COL6A2, IBSP, RAP1A and FOXF2 in NP and AF are associated with IDD progression by disrupting the extracellular matrix organization and focal adhesions pathway. In addition, IFIT1, IFIT2 and IFIT3 may negatively regulate the cell cycle, and thus decrease the number of disc cells, eventually accelerating degeneration of intervertebral discs. However, further experiments, clinical and mechanistic, are required to confirm the results of the present study.