中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2021, Vol. 16 ›› Issue (10): 2048-2055.doi: 10.4103/1673-5374.308100

• 原著:脊髓损伤修复保护与再生 • 上一篇    下一篇

脊髓损伤后环状RNA表达差异在病理进程中的作用

  

  • 出版日期:2021-10-15 发布日期:2021-03-19
  • 基金资助:

    国家自然科学基金（81874002），四川省科技支撑项目（2018SZ0159，2018SZ0246），四川省科技馆创新创业项目（2019JDRC0100）资助，国家老年临床研究中心，四川大学华西医院项目（Y2018B22，Z20192013），华西医院博士后研究发展基金（2019HXBH068）。

Role of circular RNA expression in the pathological progression after spinal cord injury

Wen-Zhao Wang#, Jun Li#, Lei Liu, Zheng-Dong Zhang, Ming-Xin Li, Qin Li, Hui-Xu Ma, Hai Yang, Xiao-Ling Hou*   

  1. Department of Orthopedics, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University/West China School of Nursing, Sichuan University, Chengdu, Sichuan Province, China
  • Online:2021-10-15 Published:2021-03-19
  • Contact: Xiao-Ling Hou, 296825257@qq.com.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, No. 81874002 (to LL); the Science and Technology Support Project of Sichuan Province of China, Nos. 2018SZ0159 (to LL), 2018SZ0246 (to XLH); the Innovation and Entrepreneurship Project of Sichuan Technology Gallery of China, Nos. 2019JDRC0100 (to JL), 2020JDRC0054 (to WZW); the National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, China, Nos. Y2018B22 (to LL), Z20192013 (to JL) and West China Hospital Postdoctoral Research and Development Fund, No. 2019HXBH068 (to JL). 

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摘要：

创伤性脊髓损伤后非编码RNA的差异表达与病理生理过程密切相关。实验系统分析和表征了创伤性脊髓损伤后脊髓组织损伤中心处环状RNA(circRNA)表达模式，并预测circRNA/miRNA调控网络的结构及潜在功能。将48只C57BL/6小鼠随机分为脊髓损伤组和假手术组。创伤性脊髓损伤组实施标准Allen’s打击T8-10节段SCI损伤，而假手术组仅行椎板切除。脊髓样品用苏木精和-伊红染色后观察，RNA测序并验证。使用荧光素酶报告基因、qRT-PCR和Western blot检测来分析创伤性脊髓损伤小鼠模型中互作网络的调控模式；①苏木精和-伊红染色显示，创伤性脊髓损伤组的脊髓组织血-脊髓屏障严重受损；②实验获取了TSCI后差异表达circRNA和miRNA的差异表达谱，细胞质中富集的差异表达circRNA参与了转录的正向调控和蛋白磷酸化；③miR-135b-5p是创伤性脊髓损伤后下调最显著的miRNA，生物信息学预测circAbca1和KLF4分别为与miR-135b-5p靶向结合的circRNA和mRNA；④实验预测并构建了circAbca1/ miR-135b-5p/KLF4调控轴,对其进行了靶向结合的验证，且抑制circAbca1后，GAP43的表达上调。⑤结果证实，环状RNA的差异表达可能在创伤性脊髓损伤后病理进程中发挥重要作用。创伤性脊髓损伤后circAbca1通过与miR-135b-5p/KLF4轴的靶向结合，起到神经抑制作用。circRNA/miRNA/mRNA网络可以为理解创伤性脊髓损伤的病理生理机制提供新的思路，为指导制定创伤性脊髓损伤的治疗策略提供新方向。

https://orcid.org/0000-0002-2403-9394 (Xiao-Ling Hou)

关键词:

脊髓损伤, 创伤, 基因, 环状RNA, circRNA/miRNA/mRNA网络, miR-135b-5p, KLF4, 生物信息学

Abstract: Differential expression of non-coding RNA after traumatic spinal cord injury (TSCI) is closely related to the pathophysiological process. The purposes of this study were to systematically profile and characterize expression of circular RNA (circRNA) in the lesion epicenter of spinal tissues after TSCI, and predict the structure and potential function of the regulatory circRNA/miRNA network. Forty-eight C57BL/6 mice were randomly and equally assigned to two groups: one subjected to TSCI at T8–10 with an Allen’s drop impactor, and a second subjected to laminectomy without TSCI. Spinal cord samples were stained with hematoxylin and eosin, sequenced, and validated. RNA-Seq, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and network analyses (Targetscan and miRanda) were used to predict and annotate the circRNA/miRNA/mRNA network. Luciferase reporter, quantitative reverse transcription polymerase chain reaction, and western blot assays were used to profile expression and regulation patterns of the network in mouse models of TSCI. Hematoxylin-eosin staining revealed severe damage to the blood-spinal cord barrier after TSCI. Differentially expressed circRNA and miRNA profiles were obtained after TSCI; differentially expressed circRNAs, which were abundant in the cytoplasm, were involved in positive regulation of transcription and protein phosphorylation. miR-135b-5p was the most significantly downregulated miRNA after TSCI; circRNAAbca1 and KLF4 were predicted to be its target circRNA and mRNA, respectively. Subsequently, the circAbca1/miR-135b-5P/KLF4 regulatory axis was predicted and constructed, and its targeted binding was verified. After inhibiting circAbca1, GAP43 expression was upregulated. Differential expression of circRNAs might play an important role after TSCI. circAbca1 plays a neuroinhibitory role by targeted binding of the miR-135b-5P/KLF4 axis. The identified circRNA/miRNA/mRNA network could provide the basis for understanding pathophysiological mechanisms underlying TSCI, as well as guide the formulation of related therapeutic strategies. All animal protocols were approved by the Research Ethics Committee of West China Hospital of China (approval No. 2017128) on May 16, 2017. 

Key words: bioinformatics, circRNA/miRNA/mRNA network, circular RNA, gene, KLF4, miR-135b-5p, spinal cord injury, trauma

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