中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2022, Vol. 17 ›› Issue (7): 1518-1525.doi: 10.4103/1673-5374.330623

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

Ezh2基因沉默可抑制背根神经节神经元轴突的再生

  

  • 出版日期:2022-07-15 发布日期:2022-01-17
  • 基金资助:

    国家重点基础研究项目2017YFA01040701（to XSG）；中国国家自然科学基金（31730031，81870975）；江苏省高等教育机构优先学术项目发展（PAPD）；江苏省自然科学基金（BK20202013）。

Silencing the enhancer of zeste homologue 2, Ezh2, represses axon regeneration of dorsal root ganglion neurons

Ting-Ting Guo1, Ying Zhao1, Wei-Xiao Huang1, Tao Zhang1, Li-Li Zhao1, 2, Xiao-Song Gu1, *, Song-Lin Zhou1, *#br#   

  1. 1Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, NMPA Key Laboratory for Research and Evaluation of Tissue Engineering Technology Products, Jiangsu Clinical Medicine Center of Tissue Engineering and Nerve Injury Repair, Co-Innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China; 2Model Animal Research Center and MOE Key Laboratory of Animal Models of Disease, Nanjing University, Nanjing, Jiangsu Province, China
  • Online:2022-07-15 Published:2022-01-17
  • Contact: Xiao-Song Gu, PhD, nervegu@ntu.edu.cn; Song-Lin Zhou, PhD, songlin.zhou@ntu.edu.cn.
  • Supported by:
    This study was supported by the National Key Basic Research Program of China, No. 2017YFA0104701 (to XSG); the National Natural Science Foundation of China, No. 31730031 (to XSG), 81870975 (to SLZ); Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) (to XSG); the Natural Science Foundation of Jiangsu Province, No. BK20202013 (to XSG).

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摘要：

与中枢神经系统不同，周围神经系统的条件性损伤会导致基因表达发生变化，发生基因重编程，进而可以诱导一系列的再生相关基因表达，最终导致损伤神经元轴突的再生。目前虽然已经发现了一些再生相关基因，但其在轴突再生下的调控机制仍不清楚。为此，实验对大鼠坐骨神经损伤后不同时间点(0 h、3 h、6 h、12 h、1 d、3 d、7 d)L4、L5背根神经节神经元进行了RNA测序，采用激光捕获显微镜技术结合NeuN标记进行神经元分离。将未损伤大鼠（正常大鼠）与坐骨神经夹伤后各组大鼠的单细胞测序结果比较，(1)见坐骨神经损伤大鼠损伤组织共发现1 228个差异表达基因。这些基因中的hub基因包括Atf3、Jun、Myc、Ngf、Fgf2、Ezh2、Gfap、IL6等；(2)随后实验证实了损伤后背根神经节神经元中的Ezh2基因的表达上调，这种上调在不同直径大小的神经元之间存在显著性差异；(3)为了证实Ezh2的上调是否影响轴突再生，实验用siRNA沉默培养的背根神经节神经元中的Ezh2基因表达后发现，此时新生轴突生长速度变慢，轴突生长的长度可受到抑制；(4)实验还预测了IPA对Ezh2的调节网络，主要发现了一些Ezh2的调节因子(Erk、Il6、Hif1a等)和靶点(Atf3、Cdkn1a、Smad1等)；(5)上述结果均说明，作为一种神经再生相关基因，Ezh2参与了坐骨神经损伤大鼠背根神经节损伤神经元的修复，体外敲除Ezh2可抑制背根神经节神经元的轴突生长。

https://orcid.org/0000-0002-2562-6275 (Xiao-Song Gu); https://orcid.org/0000-0001-8598-0922 (Song-Lin Zhou)

关键词:

背根神经节神经元, 激光捕获显微术, scRNA序列, 坐骨神经损伤, Ezh2型, IB4型, NF160/200型, RT-qPCR, siRNA, 轴突再生

Abstract: Recovery from injury to the peripheral nervous system is different from that of the central nervous system in that it can lead to gene reprogramming that can induce the expression of a series of regeneration-associated genes. This eventually leads to axonal regeneration of injured neurons. Although some regeneration-related genes have been identified, the regulatory network underlying axon regeneration remains largely unknown. To explore the regulator of axon regeneration, we performed RNA sequencing of lumbar L4 and L5 dorsal root ganglion (DRG) neurons at different time points (0, 3, 6, 12 hours, 1, 3 and 7 days) after rat sciatic nerve crush. The isolation of neurons was carried out by laser capture microscopy combined with NeuN immunofluorescence staining. We found 1228 differentially expressed genes in the injured sciatic nerve tissue. The hub genes within these differentially expressed genes include Atf3, Jun, Myc, Ngf, Fgf2, Ezh2, Gfap and Il6. We verified that the expression of the enhancer of zeste homologue 2 gene (Ezh2) was up-regulated in DRG neurons after injury, and this up-regulation differed between large- and small-sized dorsal root ganglion neurons. To investigate whether the up-regulation of Ezh2 impacts axonal regeneration, we silenced Ezh2 with siRNA in cultured DRG neurons and found that the growth of the newborn axons was repressed. In our investigation into the regulatory network of Ezh2 by interpretive phenomenal analysis, we found some regulators of Ezh2 (including Erk, Il6 and Hif1a) and targets (including Atf3, Cdkn1a and Smad1). Our findings suggest that Ezh2, as a nerve regeneration-related gene, participates in the repair of the injured DRG neurons, and knocking down the Ezh2 in vitro inhibits the axonal growth of DRG neurons. All the experimental procedures approved by the Administration Committee of Experimental Animals of Jiangsu Province of China (approval No. S20191201-201) on March 21, 2019.

Key words: axon regeneration, dorsal root ganglion neurons, Ezh2, IB4, laser capture microscopy, NF160/200, quantitative reverse transcription-polymerase chain reaction, sciatic nerve crush, scRNA-seq, siRNA