Identification of key candidate genes in local dorsal root ganglion inflammation by integrated bioinformatics analysis
- Authors:
- Published online on: June 2, 2021 https://doi.org/10.3892/etm.2021.10253
- Article Number: 821
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Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Animal chronic pain models are usually classified as peripheral nerve injury models or inflammatory pain models (1). Both nerve injury and inflammation can produce spontaneous pain, hyperalgesia and abnormal pain, and both can affect spontaneous discharges from the dorsal root ganglia (DRG) (2,3). However, the potential mechanisms of DRG discharge differ between nerve injury and chronic inflammation. Nerve injury typically leads to overall downregulation of sodium channels and altered homotype expression (4). By contrast, chronic peripheral inflammation generally leads to the upregulation of tetrodotoxin-resistant sodium channels (5). Inflammation is a factor in most pain models, including those based on nerve injury (6). Macrophage infiltration, local pro-inflammatory cytokine release (7), DRG glial cell activation and retrograde transport to DRG are important triggers of hyperalgesia (8). Inflammation also occurs under clinical pain conditions, including postherpetic neuralgia and back pain after lumbar disc herniation (9,10); the substance released from the nucleus pulposus is immunogenic, causing inflammation in adjacent DRGs. To learn more concerning the contribution of inflammation to pathological pain, Wang et al (11) developed the localized inflammation of the DRG (LID) model. In this model the cell bodies of sensory neurons are directly stimulated by the immune activator zymosan, without nerve injury. Considering the association between inflammatory processes and states of inflammatory and neuropathic chronic pain, the present study aimed to explore gene activity and expression changes induced by local inflammation of DRG.
Strong et al (12) submitted the GSE38859 dataset to the Gene Expression Omnibus (GEO) database. They screened behavior-related gene expression changes after DRG inflammation and demonstrated that immune-related genes were the largest category altered, including members of the complement system and several upregulated chemokine ligands, such as C-X-C motif ligand (CXCL)9, CXCL10 and CXCL16(12). However, their study only focused on gene function and the role of numerous differentially expressed genes (DEGs) was not explored further. In the present study, in order to identify the key candidate genes and pathway changes in DRG inflammation various bioinformatics technologies were used to reanalyze the microarray data in the GEO database. CXCL9, complement component 3 (C3), and matrix metallopeptidase 9 (MMP9) were found to have strong interactive relationships with other genes, suggesting that they may be potential targets for the treatment of DRG inflammation-induced pain. These findings may provide greater insight into the genetic mechanisms underlying DRG inflammation pain and present potential therapeutic targets for the treatment of lower back pain.
Materials and methods
Microarray data set collection and identification of DEGs
The microarray expression dataset GSE38859 was obtained from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). Exon expression profiling was based on the Agilent GPL6543 platform (Affymetrix Rat Exon 1.0 ST Array) and provided 6 sham and 6 inflamed DRG tissues. The probes were converted to the corresponding gene symbols according to the annotation information in the raw data. To reduce multiple testing, each corresponded to a unique gene symbol and only the probe set with the highest average expression was considered when multiple probe sets were associated with the same gene. Pre-treatment standardization on gene expression data for each experiment was performed using the R/Bioconductor Limma package. R is the language and operating environment for statistical analysis and drawing. DEGs were uploaded to omicstudio (https://www.omicstudio.cn/tool?order=complex), a visual analytics platform for principal component analysis. After linear model fitting, the Bayesian linear model of the limma package was estimated to identify DEGs. Statistically significant DEGs were defined with P<0.05 and |logFC|>1 as a cut-off criterion. Heatmap and volcano plots visualizations were performed using the R packages ‘pheatmap’ and ‘ggplot2’, respectively.
Enrichment analyses of DEGs
In the present study, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs were carried out using the R package. P<0.01 was chosen as the cutoff criteria. Gene Set Enrichment Analysis (GSEA) was also performed using the ‘clusterProfiler’ package in R (13), and all visualization was handled in R using the ggplot2 graphics package. The whole gene expression values of the samples were analyzed based on the h.all.v 7.0.entrez.gmt [Hallmarks] gene set database (https://www.gsea-msigdb.org/gsea/msigdb/collections.jsp#H). Significant enrichment pathways were defined by FDR <0.25 and P<0.05.
Module screening from the protein-protein interaction (PPI) network
Comprehensive information on the proteins was identified and the Search Tool for Retrieval of Interacting Genes (STRING; v11.0; https://string-db.org/), a search tool for retrieving interacting genes/proteins, was used to evaluate protein-PPI information. Interaction between proteins within a cell facilitates our understanding of how proteins operate in a coordinated manner in the cell (14). Subsequently, the PPI network was constructed and visualized by Cytoscape software (version 3.7.1; https://cytoscape.org/). Molecular Complex Detection (MCODE) analysis, an app in Cytoscape, was then used to select the most significant PPI network modules. The criteria for selection were as follows: MCODE score >3; degree cutoff, 2; node score cut-off, 0.2; and max depth, 100.
Animals and local inflammation of the DRG (LID) models
Adult male Sprague Dawley rats purchased from the Experimental Animal Center of Zhejiang Province were used in this study. Rats used for experiments were aged 6-8 weeks, weighed 250-350 g and were sex-matched. A total of 16 rats were used in this present study. A sample size of 8 rats was used per experiment to ensure repeatability. The animals were housed in a temperature-controlled animal facility (room temperature, 25˚C; humidity, 40-60%) on a 12 h light–dark cycle and food and water was freely available. All procedures were approved by the Animal Care and Use Committee of Ningbo University following the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH) (15).
An intraperitoneal injection was used to anesthetize the selected animals with sodium pentobarbital (50 mg/kg), before a longitudinal incision was made in the middle of the S1 to L4 spine. The back skin and muscular fasciae were bluntly isolated, exposing the L5 intervertebral foramen. With the needle still inside, 10 µl of the immune activator zymosan (2 mg/ml in incomplete Freund's adjuvant; Sigma-Aldrich; Merck KGaA) was slowly injected into the L5 intervertebral foramen, above the DRG. The needle remained in place for an additional 3 min after injection to avoid leakage. A control group, sham animals, experienced the same surgery process without the final step of injecting zymosan.
Reverse transcription-quantitative PCR (RT-qPCR)
RT-qPCR analysis was performed as previously described (16). After 3 days following LID- or sham- surgery, rats were decapitated following an overdose of pentobarbital sodium (150 mg/kg). Freshly isolated DRG tissues were collected on ice and immersed in TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and immediately stored at -80˚C until the time of RNA extraction. Complementary DNA (cDNA) was synthesized with the reverse transcription enzyme SuperScript II (Invitrogen; Thermo Fisher Scientific, Inc.) together with reverse transcription primers at 50˚C for 15 min and 85˚C for 5 sec. cDNA was then amplified using a HiFiScript cDNA Synthesis Kit (CoWin Biosciences) using an ABI Q5 RT-PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). All primers were synthesized by BGI Genomics Co., Ltd. (Table I). The synthesized cDNA was used qPCR to determine the expression changes in corresponding genes. The reaction mixture was: 10 µl 2X SYBR Premix Ex Taq TMII, 1 µl 10 µM forward primer, 1 µl 10 µM reverse primer, 7 µl ddH2O and 1 µl cDNA, in a total volume of 20 µl. The thermocycling conditions were: 95˚C for 2 min, followed by 40 cycles of 95˚C for 10 sec, 60˚C for 50 sec. GAPDH or Actin were used as internal reference genes, and the relative expression changes in target genes were calculated by the 2-∆∆Cq method (17).
Western blotting
The fresh tissues of mice were lysed in precooled RIPA buffer (EMD Millipore), and then homogenized using an automatic rapid sample grinder. The tissue homogenate was transferred to a sterile centrifuge tube, centrifuged at 4˚C and 10,000 x g for 10 min, and the supernatant was transferred to a new centrifuge tube. The protein concentration was determined using the BCA method (Thermo Fisher Scientific, Inc.). Total protein (20-30 µg) was separated via SDS-PAGE electrophoresis (concentrated gel, 5%; separation gel, 10-12%). After SDS-PAGE electrophoresis, the electrophoresis was carried out at a constant current of 200 mA for 2 h. At the end of membrane transfer, the nitrocellulose membrane was rinsed with TBS-0.02% Tween-20 (TBST) and sealed at room temperature for 1-2 h with blocking solution (TBST containing 3-5% skimmed milk powder). Rabbit anti-C3 (cat. no. 97425; Cell Signaling Technology, Inc.; 1:1,000), mouse anti-actin (cat. no. 3700; Cell Signaling Technology, Inc.; 1:10,000), rabbit anti- MMP9 (cat. no. 13667; Cell Signaling Technology, Inc.; 1:1000) and mouse anti-CXCL9 (cat. no. 93556; Cell Signaling Technology, Inc.; 1:1,000) were diluted with a primary antibody diluent [TBST solution containing 3% BSA (Sigma-Aldrich; Merck KGaA) and 0.01% sodium azide] and incubated at 4˚C for 15-18 h. Subsequently, the membranes were washed five times with TBST for 5 min each time. Horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG: cat. no. E030110-01; EarthOx, LLC; 1:10,000; goat anti-rabbit IgG: cat. no. E030120-01; EarthOx, LLC; 1:10,000) was diluted in TBST solution and incubated at room temperature for 2 h. Subsequently, TBST was used to rinse the membrane 5 times and TBS was used to rinse the membrane once, each time for 5 min. After rinsing, SuperSignal™ West Atto Ultimate Sensitivity ECL substrate (cat. no. A38555; Thermo Fisher Scientific, Inc.) was added and then incubated in the dark for 1-5 min, before exposure to the chemiluminescence imaging system. The protein expression detected via western blotting was quantified using ImageJ v1.8.0 (NIH).
Statistical analysis
Data are presented as the mean ± SEM. Analyses were performed using Microsoft Excel (version 16.44; Microsoft Corporation). Student's t-test was used to analyze statistical differences. P<0.05 was considered to indicate a statistically significant difference.
Results
Data normalization
The primary purpose of normalization was to eliminate technical and systematic variability from the data compared between different samples. After microarray data normalization, biological variability between different samples was assessed by plotting a principal component analysis graph (Fig. 1A). DRG-inflamed (n=6) and DRG-sham (n=6) overall had distinct, non-overlapping expression profiles. The density plot results demonstrated that the distribution of the sample intensities were generally consistent and could be used for downstream analysis (Fig. 1B). A box plot indicates each sample's gene expression and the black lines in the boxes were almost on the same straight line, indicating that the raw data were normalized successfully, which ensures the accuracy of the data (Fig. 1C).
Identification of DEGs
A total of 66 DEGs were screened, including 56 upregulated and 10 downregulated DEGs. In addition, the volcano plot of the DEGs is presented in Fig. 2A and heatmap plots in Fig. 2B.
Functional enrichment analysis of DRGs
To further investigate the function of DEGs, GO term and KEGG pathway analyses were displayed in R. DEGs were divided into three major functional categories, namely biological processes (BPs), molecular functions (MFs) and cellular components (CCs). Collectively, the data showed that DEGs were associated with GO terms related to immunity, including ‘innate immune response’, ‘activation of immune response’ and ‘activation of inflammatory response’ (Fig. 3A and Table II). In the MF category, DEGs were enriched in the terms ‘carbohydrate binding’, ‘G protein-coupled receptor binding’ and ‘heparin binding’ (Fig. 3A and Table III). In the CC analysis, the DEGs were enriched in the terms ‘external side of plasma membrane’, ‘side of membrane’ and ‘rough endoplasmic reticulum’ (Fig. 3A and Table IV). In these candidate DEGs, 14 signaling pathways were enriched in pathways in the KEGG database, including ‘cytokine-cytokine receptor interaction’, ‘viral protein interaction with cytokine and cytokine receptor’ and ‘chemokine signaling pathway' pathways (Fig. 3B and Table V). In addition, GSEA was performed for all genes on the microarray. The results of GSEA suggested that the LID model expression profiles were enriched in genes associated with the terms ‘interferon-gamma response’ and ‘inflammatory response’ (Fig. 3C).
PPI network and module analysis
To investigate interaction between the DEGs, a PPI network was constructed in STRING, which consisted of 182 edges and 62 nodes. STRING analysis showed that a total of 54 genes were filtered into the DEG PPI network complex. The network was visualized using the software tool Cytoscape (Fig. 4A). Moreover, three significant models were screened out from the PPI network by module analysis using MCODE in Cytoscape (Fig. 4A and Table VI). A total of 9 genes (MMP9, CXCL3, C3, Ptprc, CXCL11, CXCL9, CCr5, CXCL10 and C1qa) were screened out based on the high degree of connectivity (≥10; Table VI). Furthermore, MMP9, CXCL3 and C3 overlapped within the top 10 DEGs. The sham DRG on the third day was compared with the LID DRG to verify the relevance of the screened genes via RT-qPCR. This time point was selected due to the frequent spontaneous activity of the DRG. Consistent with the bioinformatics results, the expression of MMP9, C3 and CXCL9 increased significantly after LID surgery (Fig. 4D).
Discussion
In the present study, 66 DEGs were screened from GSE38859 and used for further analyses in which potential targets were identified that may be useful for the treatment and diagnosis of LID. The DRG inflammation process must be more extensively understood in order to identify the most promising genes among a large list of candidate genes. The top 10 DEGs (H2-Ea, RT1-Ba, MMP9, C3, Gbp2, Klrk1, Fcgr2b, Ifi4, CXCL9 and Lyz2) were considered the most promising candidates likely to affect the DRG inflammation process. The PPI network showed interactions among the identified DEGs. The key nodes in the network may play critical roles in the pathological process of LID. In the PPI network, 9 DEGs (MMP9, CXCL3, C3, Ptprc, CXCL11, CXCL9, CCR5, CXCL10 and C1qa) were classified as hub genes, and 3 of these were among the top 10 DEGs (CXCL9, MMP9 and C3).
As localized inflammation of the L5 DRG induced a marked increase in spontaneous bursting activity, the material was sampled for RT-qPCR on the third day (18). The qPCR and western blotting results were consistent with the bioinformatics results, showing that CXCL9, MMP9 and C3 were increased significantly in a LID model in comparison with a sham.
Chemokine-mediated neuroinflammation plays a critical role in neuropathic pain pathogenesis (19,20). CXCL9, also known as monokine induced by interferon-γ, is a CXC family chemokine (21). CXCL9 is produced by interferon-γ-stimulated macrophages and glial cells (22). A recent article reported that CXCL9 is primarily expressed in calcitonin gene-related peptide-positive and isolectin B4-positive DRG neurons and participates in the development of cancer-induced pain (23). However, a different study noted that spinal CXCL9 does not contribute to neuropathic pain despite its upregulation in the spinal cord after spinal nerve injury (24). This suggests that the mechanism of neuropathic pain differs from pain models and that CXCL9 has different functions in different tissues and distinct tissue specificity. CXCL9 was identified as a seed gene in the present PPI analysis. Seed gene are most closely related to disease genes, and these genes may become new disease-related targets. In light of these observations, it was concluded that CXCL9 may be a good candidate biomarker for diagnosing DRG inflammation.
Kawasaki et al (25) found increased MMP9 levels shortly after nerve injury in injured DRG primary sensory neurons. Moreover, treatment with an MMP9 inhibitor delayed allodynia and hyperalgesia for 11 days (26,27), implying that MMP9 participates in the onset rather than the maintenance of neuropathic pain. Liou et al (26) used a spinal nerve ligation (SNL) model to demonstrate that MMP9 concentrations were upregulated after nerve injury and then returned to the normal ranges within 14 days. MMP9 is significantly associated with the onset of neuropathic pain rather than its maintenance (28). However, specific molecular mechanisms related to MMP9 and DRG inflammation pain are lacking; this mechanism should be further investigated, as MMP9 may be a novel candidate biomarker for DRG inflammation pain.
Complement is a key component of the innate immune system, and mounting evidence suggests that ongoing complement activation may lead to pain after inflammation and injury (29). C5a and C3a can activate and sensitize skin nociceptors (30). C3 knockout rats have reduced intradermal nerve fiber density after paclitaxel treatment and reduced mechanical allodynia (31). However, the role of complement in LID remains unclear. C3 may be a potential candidate biomarker for LID.
GO and KEGG analyses of the DEGs were performed to further understand the molecular basis for DRG inflammatory pain mechanisms. GO BPs were mainly enriched in inflammation and immunity terms, including ‘response to lipopolysaccharide’, ‘response to molecule of bacterial origin’ and ‘acute inflammatory response’. KEGG pathway enrichment analysis suggested that these DEGs were related to the terms ‘chemokine signaling pathway’ and ‘cytokine-cytokine receptor interactions’.
Neuropathic pain can cause central sensitization (32,33), and its mechanisms include cytokine and chemokine release by spinal cord glial cells (19,34,35). Increasing evidence indicates that chemokine signals are crucial players in neuropathic pain (20,34,36-38). CXCL10 promotes neuropathic pain by increasing the permeability of the blood-spinal cord barrier (20). The activation of C-C chemokine receptor type 5 reduces the analgesic function of opioid receptors and enhances pain at the inflammation site (39,40).
The aforementioned findings are consistent with the KEGG analysis in the present study; however, these studies were based on spared nerve injury or SNL models. Therefore, the specific molecular mechanism of chemokines in DRG inflammation pain needs further study.
To verify the consistency of the GO and KEGG pathway enrichment analyses, GSEA analysis was performed on all genes. The analysis showed that the LID model was closely related to the terms ‘inflammatory response’, ‘interferon-γ response’ and interferon-α response pathways, supporting the GO and KEGG analyses' results.
The molecular mechanisms of lower back pain caused by DRG inflammatory pain and nerve root pain may differ. The present study's main purpose was to identify candidate genes related to local DRG inflammation, providing potential targets for the treatment or monitoring of certain forms of lower back pain that are unrelated to mechanical oppression. Although the genes identified in the present study were initially confirmed in a previous study, further studies are needed to explore these genes and pathways' specific regulatory mechanisms.
In summary, 66 DEGs were subjected to extensive bioinformatics analyses and CXCL9, MMP9, and C3 were identified as the most promising biomarkers or therapeutic targets for DRG inflammation.
Acknowledgements
Not applicable.
Funding
Funding: This study was funded by a grant from the Medical and Health Science and Technology project of Zhejiang Province (grant no. 2019KY569).
Availability of data and materials
The datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus repository, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38859.
Authors' contributions
LC, JZ and ZY contributed to animal experiments, analysis and interpretation of the data and drafted the manuscript. LC, PW, WC and YW contributed to experiments, analysis and interpretation of the data and writing of the manuscript. PW supervised the study and contributed to the conception and design of the study, the analysis and interpretation of the data and writing of the manuscript. All authors read and approved the final version of the manuscript. LC and PW confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All animal procedures were approved by the Animal Care and Use Committee of Ningbo University.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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