Zhu Y, Xu Q, Sha W-P, Zhao K-P, Wang L-M
Department of Orthopedics, Zhangjiagang Hospital Affiliated to Soochow University, Zhangjiagang, China.
Eur Rev Med Pharmacol Sci. 2019 Jan;23(1):37-43. doi: 10.26355/eurrev_201901_16745.
The aim of this study was to explore the specific role of miR-219-5p in spinal cord injury (SCI), and to investigate its underlying mechanism.
The SCI model was first constructed in mice, and the motor function of each mouse was evaluated by the Basso Beattie Bresnahan (BBB) method. The protein and mRNA expression levels of miR-219-5p and NEUROD2 in SCI mice were detected by Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), respectively. Subsequently, all mice were assigned into 3 groups, including the sham operation group, the SCI group and the SCI+miR-219-5p group. The levels of inflammatory factors (TNF-α, IL-1β and IL-6) were detected by Western blot and qRT-PCR. Meanwhile, reactive oxygen species (ROS) were detected by flow cytometry. Target genes of miR-219-5p were predicted by TargetScan and verified by Luciferase reporter gene assay. For in vitro experiments, the possible molecule mechanism of miR-219-5p in regulating NEUROD2 was detected by Western blot.
MiR-219-5p was significantly downregulated after SCI. The expression level of miR-219-5p was decreased in the SCI group than that of the sham operation group in a time-dependent manner, which reached the lowest level on the 7th day. Besides, the mRNA and protein levels of NEUROD2 in the SCI group were both remarkably increased in a time-dependent manner, which reached a peak on the 7th day. The levels of inflammatory factors (TNF-α, IL-1β and IL-6) and ROS were significantly higher in the SCI group, which could be reversed by miR-219-5p mimics transfection in SCI mice. Meanwhile, the BBB score in the SCI group was remarkably lower than that of the SCI + miR-219-5p group from the 4th day after SCI. TargetScan predicted that NEUROD2 was the target gene of miRNA-219-5p. In addition, Western blot results indicated that miR-219-5p could regulate NEUROD2, eventually promoting the recovery of SCI.
Overexpressed miR-219-5p promotes SCI recovery and motor function elevation via alleviating NEUROD2-regulated inflammation and oxidative stress.
本研究旨在探讨miR-219-5p在脊髓损伤（SCI）中的具体作用，并研究其潜在机制。
首先在小鼠中构建SCI模型，采用Basso Beattie Bresnahan（BBB）法评估每只小鼠的运动功能。分别通过蛋白质印迹法和定量实时聚合酶链反应（qRT-PCR）检测SCI小鼠中miR-219-5p和NEUROD2的蛋白质和mRNA表达水平。随后，将所有小鼠分为3组，包括假手术组、SCI组和SCI+miR-219-5p组。通过蛋白质印迹法和qRT-PCR检测炎症因子（TNF-α、IL-1β和IL-6）水平。同时，通过流式细胞术检测活性氧（ROS）。通过TargetScan预测miR-219-5p的靶基因，并通过荧光素酶报告基因测定进行验证。对于体外实验，通过蛋白质印迹法检测miR-219-5p调节NEUROD2的可能分子机制。
SCI后miR-219-5p显著下调。SCI组中miR-219-5p的表达水平随时间呈依赖性降低，在第7天达到最低水平。此外，SCI组中NEUROD2的mRNA和蛋白质水平均随时间显著升高，在第7天达到峰值。SCI组中炎症因子（TNF-α、IL-1β和IL-6）和ROS水平显著更高，在SCI小鼠中通过转染miR-219-5p模拟物可使其逆转。同时，从SCI后第4天起，SCI组的BBB评分显著低于SCI+miR-219-5p组。TargetScan预测NEUROD2是miRNA-219-5p的靶基因。此外，蛋白质印迹结果表明miR-219-5p可调节NEUROD2，最终促进SCI的恢复。
过表达的miR-219-5p通过减轻NEUROD2调节的炎症和氧化应激促进SCI恢复和运动功能提高。
