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腰椎间盘脱出症非手术治疗前后外周血基因表达变化特征及意义

戴国钢 王一 廖仕川 夏姣 王丰 黄雷 杜万里 田国刚 文江 李涛

戴国钢, 王一, 廖仕川, 等. 腰椎间盘脱出症非手术治疗前后外周血基因表达变化特征及意义[J]. 四川大学学报(医学版), 2021, 52(5): 868-876. doi: 10.12182/20210960303
引用本文: 戴国钢, 王一, 廖仕川, 等. 腰椎间盘脱出症非手术治疗前后外周血基因表达变化特征及意义[J]. 四川大学学报(医学版), 2021, 52(5): 868-876. doi: 10.12182/20210960303
DAI Guo-gang, WANG Yi, LIAO Shi-chuan, et al. Characteristics and Significance of Gene Expression Changes in Peripheral Blood of Lumbar Disc Extrusion Patients before and after Nonoperative Treatment[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2021, 52(5): 868-876. doi: 10.12182/20210960303
Citation: DAI Guo-gang, WANG Yi, LIAO Shi-chuan, et al. Characteristics and Significance of Gene Expression Changes in Peripheral Blood of Lumbar Disc Extrusion Patients before and after Nonoperative Treatment[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2021, 52(5): 868-876. doi: 10.12182/20210960303

腰椎间盘脱出症非手术治疗前后外周血基因表达变化特征及意义

doi: 10.12182/20210960303
基金项目: 四川省科技厅课题(No. 2018SZ0075)资助
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Characteristics and Significance of Gene Expression Changes in Peripheral Blood of Lumbar Disc Extrusion Patients before and after Nonoperative Treatment

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  • 摘要:   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、MPOCAMPRETNTLR5、IL1RNSLC8A1表达水平高于健康对照组(P<0.05);治疗后与治疗前比较,TLR5IL1RNSLC8A1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TLR4MMP9、MPOCAMPRETNTLR5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、IL1RNSLC8A1下调相关。
  • 图  1  腰椎间盘脱出患者治疗前和健康对照者外周血样本比较,上调的差异基因富集在以上条目中(P值最小的15个条目)

    Figure  1.  In the comparison of the peripheral blood samples of patients with LDE and those healthy controls, the up-regulated genes were enriched in the above items (15 items with the smallest P value)

    图  2  腰椎间盘脱出患者治疗前和健康对照者外周血样本比较,下调的差异表达基因富集在以上11个条目中

    Figure  2.  In the comparison of the peripheral blood samples of patients with LDE and those of healthy controls, the up-regulated genes were enriched in the above items

    图  3  差异表达基因KEGG通路富集分析结果

    Figure  3.  The results of KEGG pathway enrichment analysis for differentially expressed genes (DEGs)

    图  4  PPI网络

    Figure  4.  The PPI network

    The PPI network consisted of 77 nodes and 255 edges. The size of the node represented the centrality degree of the node. The three key sub-networks selected by the MEODE plug-in were displayed in green, purple, and yellow in the PPI network.

    图  5  腰椎间盘脱出患者治疗前后外周血TLR4、MMP9、MPOCAMPRETNTLR5、IL1RNSLC8A1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)

    GeneSequence (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.
    下载: 导出CSV

    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

    GenePFold changeFunction
    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
    0.02
    2.84
    Extracellular matrix structural constituent, protein binding, extracellular matrix structural constituent conferring tensile strength
     BATF2
    0.02
    2.81
    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
    <0.001
    0.63
    Retinal dehydrogenase activity, NADP+1-oxidoreductase activity, aldo-keto reductase (NADP) activity, retinol dehydrogenase activity
     KLRF1
    <0.001
    0.62
    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
     KRT86 <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
    下载: 导出CSV

    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 symbolDescriptionBefore treating vs. controlAfter treating vs. before teating
    P Fold changePFold 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.
    下载: 导出CSV

    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 symbolCentrality degreeMCODE_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
    下载: 导出CSV
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  • 收稿日期:  2020-09-14
  • 修回日期:  2021-08-03
  • 网络出版日期:  2021-12-06
  • 刊出日期:  2021-09-20

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