2120 MOLECULAR MEDICINE REPORTS 15: 2120-2128, 2017 Gene expression profiles reveal key pathways and genes associated with neuropathic pain in patients with spinal cord injury 1 1 2 3 1 XIJING HE , LIYING FAN , ZHONGHENG WU , JIAXUAN HE and BIN CHENG Departments of 1Orthopedics, 2Rehabilitation and 3Anesthesiology, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710004, P.R. China Received December 30, 2015; Accepted December 8, 2016 DOI: 10.3892/mmr.2017.6231 Abstract. Previous gene expression profiling studies of Introduction neuropathic pain (NP) following spinal cord injury (SCI) have predominantly been performed in animal models. The present Spinal cord injury (SCI) is an ever-increasing challenge and study aimed to investigate gene alterations in patients with a devastating neurologic event affecting patients and their spinal cord injury and to further examine the mechanisms families (1). A myriad of neurologic and medical sequelae can underlying NP following SCI. The GSE69901 gene expres- occur subsequent to the original insult, including the long-term sion profile was downloaded from the public Gene Expression loss of sensory and motor functions, amongst other complica- Omnibus database. Samples of peripheral blood mononuclear tions (1). Chronic pain is an important problem following SCI cells (PBMCs) derived from 12 patients with intractable NP and is a major impediment to effective rehabilitation (2). It and 13 control patients without pain were analyzed to iden- has been shown that chronic pain following SCI has a high tify the differentially expressed genes (DEGs), followed by prevalence of at least 80%, which in at least 40% of individuals functional enrichment analysis and protein-protein interac- manifests as persistent neuropathic pain (NP) (3). tion (PPI) network construction. In addition, a transcriptional NP can be categorized as peripheral and central pain, and regulation network was constructed and functional gene is often described as paroxysmal, stabbing, burning, pulsing, clustering was performed. A total of 70 upregulated and electric shock-like, pricking or tingling, and a spontaneous 61 downregulated DEGs were identified in the PBMC samples or evoked unpleasant abnormal sensation (4). Current treat- from patients with NP. The upregulated and downregulated ments use a variety of surgical, pharmacological, physical and genes were significantly involved in different Gene Ontology psychological strategies for the management of NP following terms and pathways, including focal adhesion, T cell receptor SCI (2). However, evidence of effective therapeutic approaches signaling pathway and mitochondrial function. Glycogen in use remains to be fully elucidated. Therefore, an improved synthase kinase 3 β (GSK3B) was identified as a hub protein understanding of the mechanisms underlying NP in patients in the PPI network. In addition, ornithine decarboxylase 1 with SCI and the identification of effective treatment strategies (ODC1) and ornithine aminotransferase (OAT) were regulated are required. by additional transcription factors in the regulation network. In previous years, the identification of specific molecular GSK3B, OAT and ODC1 were significantly enriched in alterations involved in NP syndromes has become a major priority two functional gene clusters, the function of mitochondrial in investigations of SCI (5). Several biological alterations have membrane and DNA binding. Focal adhesion and the T cell been implicated in the mechanisms of NP, including cellular receptor signaling pathway may be significantly linked with interactions, ion channel expression, extracellular proteins and N P, a nd GSK3B, OAT and ODC1 may be potential targets for epigenetic effects (6). For example, N-type voltage-dependent the treatment of NP. Ca2+ channels, which are expressed in non-excitable microg- lial cells in mice, have been demonstrated to contribute to the pathophysiology of NP (7). In addition, Chen et al (8) demonstrated that astrocytic connexin-43 enhances spinal cord synaptic transmission and maintains late-phase NP in Correspondence to: Dr Xijing He, Department of Orthopedics, mice via the release of chemokines. Nesic et al (9) performed Second Affiliated Hospital of Xi'an Jiaotong University, 157 Xiwu DNA microarray analysis and showed that a number of genes Road, Xincheng, Xi'an, Shaanxi 710004, P.R. China E-mail: [email protected] with increased expression were significantly associated with astrocytic activation and inflammation in the spinal cords of Key words: spinal cord injury, neuropathic pain, functional rats, which developed central NP. Vicuña et al (10) revealed enrichment analysis, protein-protein interaction network, gene that the serine protease inhibitor, serpinA3N can attenuate NP clustering by inhibiting T cell-derived leukocyte elastase in mice, and demonstrated crosstalk between T cells and neurons in the modulation of NP. However, investigations of NP following SCI have predominantly been performed in animal models HE et al: GENE EXPRESSION PROFILES OF NEUROPATHIC PAIN 2121 and the exact molecular mechanisms of persistent NP remain bioc/html/preprocessCore.htmlurisimplehttp://bioconductor. to be elucidated. org/packages/release/bioc/html/preprocessCore.html) (17). Microarray data provide a global assessment of gene A t-test in the limma package (version 3.22.7, http://www expression signatures, which may provide insights into the .bioconductor.org/packages/3.0/bioc/html/limma.html) was pathophysiology of disease (11). Previous gene expression performed to identify DEGs in the specimens from the profiling studies of NP following SCI have been performed patients with NP, compared with the controls. An absolute predominantly in animal models (12,13). However, the gene value of log2-fold change (log2FC)>1 and adjusted P-value expression profiling of NP in human whole blood has not of <0.05 were used to determine significant differential been reported. In the present study, the microarray data expression. of GSE69901 was downloaded from the publicly available Gene Expression Omnibus (GEO) database and analyzed. Functional enrichment analysis. TargetMine is an integrated Differentially expressed genes (DEGs) were screened in the database enabling complicated searches, which are difficult peripheral blood mononuclear cells (PBMCs) of samples to perform using existing comparable tools and assists in from patients with SCI and intractable NP, and compared efficient target prioritization (18). In order to analyze the with those from patients with SCI without pain. This was identified upregulated and downregulated genes at functional followed by functional enrichment analysis and construc- levels, Gene Ontology (GO) and Kyoto Encyclopedia of Genes tion of a protein-protein interaction (PPI) network. A and Genomes (KEGG) pathway enrichment analyses were transcriptional regulation network was also constructed performed using TargetMine (http://targetmine.nibio.go.jp/). and functional gene clustering was performed. The aim of GO terms or pathways with a P-value <0.05 were considered the present study was to further investigate the molecular to be significantly enriched. mechanisms underlying NP following SCI, and to identify additional potential pathways and genes associated with the PPI network construction. The online database resource pathogenesis of NP. Search Tool for the Retrieval of Interacting Genes (STRING) provides uniquely comprehensive coverage, and access to Materials and methods predicted and experimental interaction information (19). Interactions in the STRING database are provided with a Microarray data. The GEO (http://www.ncbi.nlm.nih.gov/geo/) confidence score (19). In the present study, application of the is an international public repository, which archives and freely STRING database (http://string-db.org/) was used to predict distributes high-throughput microarray and next-generation PPIs based on a confidence score >0.4 and other default sequencing functional genomic data deposited by the scientific parameters. The PPI network was visualized using Cytoscape community (14). In addition to serving as a public archive, the (version 2.8.2, http://www.cytoscape.org/) (20). GEO database provides available tools to assist users in iden- tifying, analyzing and visualizing data associated with their Transcriptional regulation network construction. The specific interests (14). In the present study, the GSE69901 micro- Human Transcriptional Regulation Interactions database array data, deposited by Adıgüzel et al on 15th June 2015, was (HTRIdb) is an open-access database (http://www.lbbc.ibb. retrieved from the publicly available GEO database (https://www. unesp.br/htri) from which human experimentally validated ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69901). As shown interactions among transcription factors (TFs) and their corre- in the description of the GSE69901 series in the GEO database, sponding target genes can be extracted. These can be used the PBMCs were collected from whole blood samples from to construct transcriptional regulation interaction networks, 12 patients with intractable NP and 13 patients in the control enabling detailed understanding of the regulation of biological group (without pain). All patients had complete SCI with a processes (21). In the present study, the TFs involved in regu- level of injury above T5. Data were generated using the plat- lating all DEGs were screened based on the data derived from form of the GPL15207 (PrimeView)