Characteristics and Significance of Gene Expression Changes in Peripheral Blood of Lumbar Disc Extrusion Patients before and after Nonoperative Treatment
目的 研究腰椎间盘脱出患者外周血基因表达特征及非手术治疗对其表达的影响。 方法 采用基因芯片半定量测定初步筛选腰椎间盘脱出患者和健康对照者外周血中的差异表达基因，以及患者在非手术治疗后这些差异表达基因的变化趋势，通过富集分析研究差异表达基因的功能特征，通过网络分析找出基因异常表达的关键基因，采用qRT-PCR定量检测验证这些基因在患者和健康对照样本中的表达情况以及非手术治疗对这些差异表达基因的影响。 结果 在腰椎间盘脱出患者和健康对照组的外周血中发现153个差异表达基因，其中131个基因表达上调，22个基因表达下调；富集分析显示大部分差异表达基因与免疫以及炎症反应相关；网络分析显示Toll样受体4（toll-like receptor 4, TLR4）、基质金属肽酶9（matrix metallopeptidase 9, MMP9）、髓过氧化物酶（myeloperoxidase, MPO)、抗菌肽（cathelicidin antimicrobial peptide, CAMP）、resistin基因（RETN）和Toll样受体5（toll-like receptor 5, TLR5）是蛋白互作网络中的关键基因，这些关键基因均被富集到了免疫、炎症反应相关的条目。非手术治疗后，患者疼痛减轻，在这153个差异表达基因中，TLR5、白介素1受体拮抗剂（interleukin 1 receptor antagonist, IL1RN）和溶质载体家族8成员A1（solute carrier family 8 member A1, SLC8A1）在治疗后表达下调。qRT-PCR结果显示：患者外周血中TLR4、MMP9、MPO、CAMP、RETN、TLR5、IL1RN和SLC8A1 表达水平高于健康对照组（P<0.05）；治疗后与治疗前比较，TLR5 、IL1RN和SLC8A1 表达水平降低（P<0.05）。 结论 腰椎间盘脱出患者外周血基因表达特征主要是免疫和炎症反应相关基因表达失调，其中TLR4 、MMP9、MPO、CAMP、RETN和TLR5这些与免疫和炎症反应相关的基因在腰椎间盘脱出患者外周血基因表达失调中起关键作用，非手术治疗疗效的获得可能与患者外周血中过度表达的TLR5、IL1RN和SLC8A1下调相关。Abstract: Objective To define the gene expression characteristics in the peripheral blood of patients with lumbar disc extrusion (LDE) and the effect of nonoperative treatment on the gene expression. Methods DNA microarray was used to identify semi-quantitatively the differentially expressed genes (DEGs) in the peripheral blood of patients with LDE and that of the healthy controls and the variation trend of these DEGs after nonoperative treatment. Enrichment analysis was done to reveal the functional characteristics of these DEGs, and network analysis was done to identify key genes that contribute to gene dysregulation. The levels of these key genes were measured by qRT-PCR to examine their expression in LDE patients and the controls, and the effect of nonoperative treatment on the expression level. Results We identified 153 DEGs in the peripheral blood of LDE patients and healthy controls, including 131 upregulated genes and 22 downregulated genes. Enrichment analysis revealed that most of the DEGs were related to immunity and the inflammatory response. Network analysis revealed that toll-like receptor 4 (TLR4 ), matrix metallopeptidase 9 (MMP9) and myeloperoxidase (MPO), cathelicidin antimicrobial peptide (CAMP), resistin (RETN), toll-like receptor 5 (TLR5) were the key genes in the protein-protein interaction network. These key genes were all enriched into the terms releated to immunity and the inflammatory response. The patients experienced pain relief after nonoperative treatment. Among the 153 DEGs, TLR5 , interleukin 1 receptor antagonist (IL1RN) and solute carrier family 8 member A1 (SLC8A1) were downregulated after nonoperative treatment. qRT-PCR revealed that the levels of TLR4, MMP9 , MPO, CAMP, RETN, TLR5, IL1RN and SLC8A1 in the peripheral blood of the LDE patients were higher than those of the healthy control group (P<0.05). In addition, TLR5, IL1RN and SLC8A1 expression levels decreased after treatmentin in comparison with the levels before treatment (P<0.05). Conclusion Gene expression in the peripheral blood of LDE patients was characterized by the dysregulation of immune and inflammatory response-related genes, among which, TLR4, MMP9, MPO, CAMP, RETN and TLR5, the genes relevant to immune and inflammatory response, played a key role in the dysregulation of gene expression in the peripheral blood of LDE patients. The outcome of non-operative treatment may be related to the downregulation of the overexpressed TLR5, IL1RN and SLC8A1 in the peripheral blood of patients.
图 5 腰椎间盘脱出患者治疗前后外周血TLR4、MMP9、MPO、CAMP、RETN、TLR5、IL1RN和SLC8A1基因表达结果
Figure 5. The expression levels of TLR4, MMP9, MPO, CAMP, RETN, TLR5, IL1RN and SLC8A1 in the peripheral blood of LDE patients before and after treatment
Control: Healthy volunteers; Baseline: Patients with LDE before nonoperative treatment; TCM: Patients with LDE after traditional Chinese medicine nonoperative treatment. n=25, * P<0.05.
表 1 实时荧光定量PCR引物序列
Table 1. Sequences of primers used for quantitative real-time polymerase chain reaction (qRT-PCR)
Gene Sequence (5′ to 3′) TLR4 F: CCTGAGGCATTTAGGCAGCTA R: GATAAATCCAGCACCTGCAGTTC MMP9 F: CACGCACGACGTCTTCCA R: AAGCGGTCCTGGCAGAAAT MPO F: CGGTACCCAGTTCAGGAAGCT R: CCCTCGTTCTCCCACCAAA CAMP F: TCAAGGATTTTTTGCGGAATCT R: GCCAGGGTAGGGCACACA RETN F: AGCCATCAATGAGAGGATCCA R: AGGCCAATGCTGCTTATTGC TLR5 F: TCTGCTAGGACAACGAGGATCA R: CCATGAGCACCACTCCTAGGA IL1RN F: CAGCTGGAGGCAGTTAACATCA R: GAAGCGCTTGTCCTGCTTTC SLC8A1 F: CCAGACACATTTGCCAGCAA R: CTATGGAGGCGTCTGCATACTG β-actin F: CTGGAACGGTGAAGGTGACA R: CGGCCACATTGTGAACTTTG F: Forward; R: Reverse.
表 2 腰椎间盘脱出患者和健康对照者外周血样本之间表达上调和下调差异倍数最大的10个差异表达基因
Table 2. The 10 differentially expressed genes (DEGs) with the largest fold change of up-regulation and down-regulation between peripheral blood samples of patients with lumbar disc extrusion (LDE) and the control group
Gene P Fold change Function Up-regulated PRDM8 0.01 3.56 DNA binding, protein binding, methyltransferase activity, transferase activity, metal ion binding. OLFM4 0.03 3.15 Catalytic activity, structural molecule activity, protein binding, cadherin binding. RPGRIP1 0.03 3.00 Protein binding, extracellular matrix structural, constituent, protein binding, DNA binding COL9A2
Extracellular matrix structural constituent, protein binding, extracellular matrix structural constituent conferring tensile strength BATF2
RNA polymerase Ⅱ proximal promoter sequence-specific DNA binding, DNA-binding transcription factor activity, RNA polymerase Ⅱ-specific FCGR1A <0.001 2.53 Transmembrane signaling receptor activity, protein binding, IgG binding CEACAM8 0.02 2.42 Protein binding, protein heterodimerization activity LTF <0.001 2.41 Lipopolysaccharide binding, DNA binding, serine-type endopeptidase activity, iron ion binding LCN2 0.02 2.24 Protease binding, iron ion binding, protein binding, small molecule binding, identical protein binding DEFA4 0.02 2.15 Protein homodimerization activity Down-regulated PLXDC1 0.01 0.63 Protein binding CD160 <0.001 0.63 Transmembrane signaling receptor activity, signaling receptor binding, protein binding, kinase binding KLRC3 <0.001 0.63 Transmembrane signaling rceptor activity, carbohydrate binding AKR1C3
Retinal dehydrogenase activity, NADP+1-oxidoreductase activity, aldo-keto reductase (NADP) activity, retinol dehydrogenase activity KLRF1
Transmembrane signaling receptor activity, protein binding, carbohydrate binding, MHC class Ⅰ receptor activity KLRB1 <0.001 0.62 Transmembrane signaling receptor activity, protein binding, carbohydrate binding KRT8 6 <0.001 0.62 Protein binding LEPROTL1 <0.001 0.61 Protein binding, identical protein binding FCGBP <0.001 0.60 Protein binding ADAMTS10 0.01 0.52 Metalloendopeptidase activity, protein binding, peptidase activity, metallopeptidase activity IGJ 0.04 0.52 Single-stranded DNA binding, antigen binding, IgA binding, protein binding, phosphatidylcholine binding
表 3 患者治疗前和对照组比较、患者治疗后和治疗前比较外周血中的差异表达基因
Table 3. DEGs findings of comparison between peripheral blood of patients before treatment and that of the controls, and DEGs findings of comparison between the peripheral blood of patients after treatment with that of patients before treatment
Gene symbol Description Before treating vs. control After treating vs. before teating P Fold change P Fold change TLR5 Toll-like receptor 5 <0.001 1.55 0.03 0.67 IL1RN Interleukin 1 receptor antagonist 0.01 1.66 0.03 0.64 SLC8A1 Solute carrier family 8 member A1 <0.001 1.48 0.04 0.63 RBM20 RNA binding motif protein 20 >0.05 − 0.03 0.62 GPER1 G protein-coupled estrogen receptor 1 >0.05 − 0.00 0.62 IL27 Interleukin 27 >0.05 − 0.01 0.59 SOCS1 Suppressor of cytokine signalling 1 >0.05 − 0.02 0.56 GRTP1-AS1 GRTP1 antisense RNA 1 >0.05 − 0.04 0.48 153 differentially expressed genes were identified in the comparison of peripheral blood of the patients before treatment and that of the control group. 8 differentially expressed genes were identified in the comparison of peripheral blood of the patients before treatment and that after treatment. The intersection of the two groups of differentially expressed genes included TLR5, IL1RN and SLC8A1.
表 4 PPI网络中具有最高中心度数的15个基因及其是否被包含在MCODE子网络中
Table 4. The 15 genes with the highest centrality degree in the PPI network and whether they were included in the MCODE sub-network
Gene symbol Centrality degree MCODE_Cluster TLR4 26 Cluster 1 MMP9 22 Cluster 2 MPO 20 Cluster 2 CAMP 18 Cluster 2 RETN 18 Cluster 1 TLR5 17 Unclustered CEACAM8 16 Cluster 1 CD86 16 Cluster 2 LCN2 15 Cluster 1 IL1RN 13 Cluster 1 PGLYRP1 13 Cluster 1 LTF 13 Cluster 1 SOCS3 12 Unclustered CTSG 11 Cluster 1 STAT1 11 Cluster 2
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