Wang Wen-Zhao, Li Jun, Liu Lei, Zhang Zheng-Dong, Li Ming-Xin, Li Qin, Ma Hui-Xu, Yang Hai, Hou Xiao-Ling
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.
Neural Regen Res. 2021 Oct;16(10):2048-2055. doi: 10.4103/1673-5374.308100.
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.
创伤性脊髓损伤（TSCI）后非编码RNA的差异表达与病理生理过程密切相关。本研究的目的是系统地分析和表征TSCI后脊髓组织损伤中心环状RNA（circRNA）的表达，并预测调控circRNA/miRNA网络的结构和潜在功能。48只C57BL/6小鼠被随机等分为两组：一组用Allen打击器在T8-10水平造成TSCI，另一组进行无TSCI的椎板切除术。对脊髓样本进行苏木精-伊红染色、测序和验证。采用RNA测序、基因本体分析、京都基因与基因组百科全书分析以及网络分析（Targetscan和miRanda）来预测和注释circRNA/miRNA/mRNA网络。利用荧光素酶报告基因、定量逆转录聚合酶链反应和蛋白质印迹分析来分析TSCI小鼠模型中该网络的表达和调控模式。苏木精-伊红染色显示TSCI后血脊髓屏障严重受损。TSCI后获得了差异表达的circRNA和miRNA谱；差异表达的circRNA在细胞质中丰富，参与转录的正调控和蛋白质磷酸化。miR-135b-5p是TSCI后下调最显著的miRNA；circRNAAbca1和KLF4分别被预测为其靶circRNA和mRNA。随后，预测并构建了circAbca1/miR-135b-5P/KLF4调控轴，并验证了其靶向结合。抑制circAbca1后，GAP43表达上调。circRNA的差异表达可能在TSCI后发挥重要作用。circAbca1通过靶向结合miR-135b-5P/KLF4轴发挥神经抑制作用。所鉴定的circRNA/miRNA/mRNA网络可为理解TSCI的病理生理机制提供依据，并指导相关治疗策略的制定。所有动物实验方案均于2017年5月16日获得中国西部医院研究伦理委员会批准（批准号：2017128）。
