南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (6): 883-890.doi: 10.12122/j.issn.1673-4254.2021.06.11

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基于转录组测序技术的椎间盘退变特异基因表达谱分析

夏博文，邢军超，艾秋池，李瀚卿，徐美涛，侯天勇   

  • 出版日期:2021-06-20 发布日期:2021-07-05

Expression profile of intervertebral disc degeneration-specific genes: a transcriptome sequencing-based analysis

  • Online:2021-06-20 Published:2021-07-05

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摘要： 目的 通过筛选分析椎间盘退变过程中的表达变化基因寻找临床治疗间盘退变的新靶点。方法 运用转录组测序技术评估了临床采集到的椎间盘退变患者（IDD，n=4）和非IDD（n=3）椎间盘样本，筛选出具有显著性的差异基因并利用基因本体论(GO）数据库和京都基因和基因组百科全书(KEGG)数据库进行基因功能富集，筛选在IDD进展中可能起关键作用的基因和信号通路，最后使用qRT-PCR技术对上述识别到的部分显著差异基因进行验证。结果 测序结果识别出512个显著差异基因（DEGs），GO数据库主要将这些DEGs主要富集到角化、细胞外基质（ECM）构成、生长因子结合以及炎症趋化作用，而KEGG富集分析的前10通路分别为：阿米巴病、病毒蛋白与细胞因子及其受体的相互作用、ECM受体的相互作用、IL-17信号通路、细胞因子与其受体的相互作用、TNF信号通路、糖尿病并发症中的AGE-RAGE信号通路、PI3K-Akt信号通路、趋化因子信号通路以及雌激素信号通路。选取的 13个作为qRT-PCR验证目标的DEGs在两组间表达具有差异显著性（P<0.001），且所有的DEGs的表达趋势均与RNA-seq结果一致，其中3个基因组间差异倍数不显著（Log2FoldChange≤1），其余10个基因具有差异显著性（Log2FoldChange>1）。结论 ECM、生长因子、胶原纤维蛋白组分、炎症趋化因子以及TNF-α和PI3K-Akt信号通路在IDD进展过程中发挥重要作用，这些因素可能成为椎间盘退变临床治疗的新靶点。

关键词: 转录组测序；椎间盘退变；差异基因；富集分析；信使RNA

Abstract: Objective To identify new therapeutic targets for intervertebral disc degeneration (IDD) by analyzing gene variations in IDD. Methods We analyzed surgical samples of intervertebral disc from 4 patients with IDD and 3 patients with non- IDD using RNA sequencing (RNA-seq) technology to identify significant differentially expressed genes (DEGs) in IDD. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were utilized for gene enrichment studies to acquire the key genes and signal pathways during IDD progression. The differential expressions of the identified genes in IDD were validated in clinical samples with qRT-PCR. Results The transcriptome profile revealed 512 significant DEGs, which were enriched in terms of keratinization, extracellular matrix (ECM) components, growth factor binding, and inflammatory chemotaxis in GO analysis. The top 10 terms of KEGG enrichment included amoebiasis, viral protein interaction with cytokine and cytokine receptor, ECM-receptor interaction, IL-17 signaling pathway, cytokine-cytokine receptor interaction, TNF signaling pathway, AGE-RAGE signaling pathway in diabetic complications, PI3K-Akt signaling pathway, chemokine signaling pathway and estrogen signaling pathway. Thirteen DEGs selected as the targets for qRT-PCR validation showed significant differential expressions in IDD (P<0.001), and their expression trends were all consistent with the results of RNA-seq. Among these genes, 10 genes showed significant intergroup fold change (Log2FoldChange>1). Conclusion ECM, growth factors, collagen components, inflammatory chemokines and such signal pathways as TNF-α and PI3K-Akt all have important contributions to IDD progression and may thus serve as new therapeutic targets for treatment of IDD.

Key words: RNA sequencing; intervertebral disc degeneration; different expressed genes; enrichment analysis; mRNA