中国神经再生研究(英文版) ›› 2023, Vol. 18 ›› Issue (9): 2056-2066.doi: 10.4103/1673-5374.366494

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

背根神经节神经元轴突再生过程中的基因表达谱:联合激光显微切割和深度测序鉴定

  

  • 出版日期:2023-09-15 发布日期:2023-03-07
  • 基金资助:
    国家自然科学基金项目(31730031,32130060);国家重大研发计划项目(No.2017YFA0104700);江苏省自然科学基金项目(BK20202013)

Transcriptional regulatory network during axonal regeneration of dorsal root ganglion neurons: laser-capture microdissection and deep sequencing

Li-Li Zhao1, 2, Tao Zhang2, 3, Wei-Xiao Huang2, 3, Ting-Ting Guo2, Xiao-Song Gu1, 2, *   

  1. 1Model Animal Research Center and MOE Key Laboratory of Animal Models of Disease, Nanjing University, Nanjing, Jiangsu Province, China; 2Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China; 3School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, Nanjing, Jiangsu Province, China
  • Online:2023-09-15 Published:2023-03-07
  • Contact: Xiao-Song Gu, PhD, nervegu@ntu.edu.cn.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, Nos. 31730031 and 32130060; the National Major Project of Research and Development, No. 2017YFA0104700; and the Natural Science Foundation of Jiangsu Province, No. BK20202013 (all to XSG).

摘要:

由于高通量的测序技术中投入的细胞数量及大小、测序的深度、细胞分离方法等都会影响最终的测序结果,因此,对于背根神经节神经元轴突再生过程中详尽的分子表达谱的研究仍有欠缺。因此实验采用了激光显微切割和深度测序联合方法对坐骨神经损伤后0,3,6,12 h,以及1,3,7 d时背根神经节神经元进行RNA测序。首先基于细胞的分子表达差异,发现背根神经节神经元在损伤后呈现了3个阶段:早期阶段(3-12h),预再生阶段(1d)以及再生阶段(3-7d)。进一步基因表达模式和相关功能富集分析发现,其中1个基因模块与轴突再生具有高度相关。继而验证了损伤后背根神经节神经元中Atf3,Klf6,Arid5a,Crem和Fosl1基因表达上调。抑制Crem,Arid5a,Fosl1和Klf6转录因子可在体外抑制神经元突起的生长。作为枢纽转录因子,Atf3在再生前和再生阶段具有较高的表达和活性,而Gper1,Il12a,ESR1和IL6 可能会是早期阶段轴突损伤修复过程中触发Atf3的激活的上游因子。这些结果揭示了周围神经损伤后背根神经节神经元轴突再生过程中详尽的分子表达谱,可为临床上筛选周围神经损伤治疗的分子靶点提供了的参考。

https://orcid.org/0000-0002-9962-9946 (Li-Li Zhao); https://orcid.org/0000-0002-2562-6275 (Xiao-Song Gu)

关键词: 背根神经节, 神经元, 激光显微切割, Smart-seq2, 基因表达谱, 转录因子, Crem, Arid5a, Fosl1, Klf6, Atf3

Abstract: The key regulators and regeneration-associated genes involved in axonal regeneration of neurons after injury have not been clarified. In high-throughput sequencing, various factors influence the final sequencing results, including the number and size of cells, the depth of sequencing, and the method of cell separation. There is still a lack of research on the detailed molecular expression profile during the regeneration of dorsal root ganglion neuron axon. In this study, we performed laser-capture microdissection coupled with RNA sequencing on dorsal root ganglion neurons at 0, 3, 6, and 12 hours and 1, 3, and 7 days after sciatic nerve crush in rats. We identified three stages after dorsal root ganglion injury: early (3–12 hours), pre-regeneration (1 day), and regeneration (3–7 days). Gene expression patterns and related function enrichment results showed that one module of genes was highly related to axonal regeneration. We verified the up-regulation of activating transcription factor 3 (Atf3), Kruppel like factor 6 (Klf6), AT-rich interaction domain 5A (Arid5a), CAMP responsive element modulator (Crem), and FOS like 1, AP-1 transcription factor Subunit (Fosl1) in dorsal root ganglion neurons after injury. Suppressing these transcription factors (Crem, Arid5a, Fosl1 and Klf6) reduced axonal regrowth in vitro. As the hub transcription factor, Atf3 showed higher expression and activity at the pre-regeneration and regeneration stages. G protein-coupled estrogen receptor 1 (Gper1), interleukin 12a (Il12a), estrogen receptor 1 (ESR1), and interleukin 6 (IL6) may be upstream factors that trigger the activation of Atf3 during the repair of axon injury in the early stage. Our study presents the detailed molecular expression profile during axonal regeneration of dorsal root ganglion neurons after peripheral nerve injury. These findings may provide reference for the clinical screening of molecular targets for the treatment of peripheral nerve injury.

Key words: Arid5a, Atf3, Crem, dorsal root ganglion, Fosl1, Klf6, laser-capture microdissection, neuron, smart-seq2, gene expression profile, transcription factor