Y Zhu1, Q Xu, W-P Sha, K-P Zhao, L-M Wang. 1. Department of Orthopedics, Zhangjiagang Hospital Affiliated to Soochow University, Zhangjiagang, China. 153472108@qq.com.
Abstract
OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: Overexpressed miR-219-5p promotes SCI recovery and motor function elevation via alleviating NEUROD2-regulated inflammation and oxidative stress.
OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: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. CONCLUSIONS: Overexpressed miR-219-5p promotes SCI recovery and motor function elevation via alleviating NEUROD2-regulated inflammation and oxidative stress.