Supplementary Material for: Circ-Ctnnb1 regulates neuronal injury in spinal cord injury through Wnt/β-catenin signaling pathway

Study design: Spinal cord injury (SCI) rat model and cell model were established for in vivo and in vitro experiments. Functional assays were utilized to explore the role of the circRNAs derived from catenin beta 1 (mmu_circ_0001859, circ-Ctnnb1 herein) in regulating neuronal cell viability and apoptosis. Bioinformatics analysis and mechanism experiments were conducted to assess the underlying molecular mechanism of circ-Ctnnb1. Objective: We aimed to probe into the biological function of circ-Ctnnb1 in neuronal cells of SCI. Methods: The rat model of SCI and hypoxia-induced cell model were constructed to examine circ-Ctnnb1 expression in SCI through quantitative reverse transcription real-time polymerase chain reaction (RT-qPCR). Basso, Beattie and Bresnahan (BBB) score was utilized for evaluating the neurological function. Terminal-deoxynucleoitidyl Transferase Mediated Nick End labeling (TUNEL) assays were performed to assess the apoptosis of neuronal cells. RNase R and Actinomycin D (ActD) were used to treat cells to evaluate the stability of circ-Ctnnb1. Results: Circ-Ctnnb1 was highly expressed in SCI rat models and hypoxia-induced neuronal cells, and its deletion elevated the apoptosis rate of hypoxia-induced neuronal cells. Furthermore, circ-Ctnnb1 activated the Wnt/β-catenin signaling pathway via sponging mircoRNA-205-5p (miR-205-5p) to up-regulate Ctnnb1 and Wnt family member 2B (Wnt2b). Conclusion: Circ-Ctnnb1 promotes SCI through regulating Wnt/β-catenin signaling via modulating the miR-205-5p/Ctnnb1/Wnt2b axis.

研究设计：构建脊髓损伤（Spinal cord injury, SCI）大鼠模型与细胞模型，开展体内外实验。通过功能实验探究β-连环蛋白（catenin beta 1）来源的环状RNA（circRNAs，本研究中记为mmu_circ_0001859，即circ-Ctnnb1）在调控神经元细胞活力与凋亡中的作用，并通过生物信息学分析与机制实验解析circ-Ctnnb1潜在的分子调控机制。
研究目的：本研究旨在探讨circ-Ctnnb1在SCI神经元细胞中的生物学功能。
实验方法：构建脊髓损伤大鼠模型与缺氧诱导细胞模型，通过实时定量反转录聚合酶链反应（quantitative reverse transcription real-time polymerase chain reaction, RT-qPCR）检测circ-Ctnnb1在脊髓损伤中的表达水平。采用Basso、Beattie与Bresnahan评分（BBB评分）评估大鼠神经功能。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定（Terminal-deoxynucleoitidyl Transferase Mediated Nick End labeling, TUNEL）检测神经元细胞凋亡情况。使用RNase R与放线菌素D（Actinomycin D, ActD）处理细胞，以评估circ-Ctnnb1的分子稳定性。
实验结果：circ-Ctnnb1在脊髓损伤大鼠模型与缺氧诱导神经元细胞中呈高表达状态；敲低circ-Ctnnb1可显著提升缺氧诱导神经元细胞的凋亡率。此外，circ-Ctnnb1可通过海绵吸附微小RNA-205-5p（mircoRNA-205-5p, miR-205-5p），上调Ctnnb1与Wnt家族成员2B（Wnt family member 2B, Wnt2b）的表达，进而激活Wnt/β-连环蛋白信号通路。
研究结论：circ-Ctnnb1通过调控miR-205-5p/Ctnnb1/Wnt2b轴介导Wnt/β-连环蛋白信号通路，进而促进脊髓损伤的发生发展。