miR-124-3p alleviates neuronal apoptosis in spinal cord injury by targeting calpain 1 to inhibit oxidative stress

English Keywords:Spinal cord injury  MiR-124-3p  Calpain 1  Apoptosis  Neurons

English Abstract:

  【Abstract】 Objectives: To investigate the effect of microRNA(miR)-124-3p on spinal cord injury and its molecular mechanism. Methods: Forty adult male Sprague-Dawley rats were randomly divided into group A(group miR-124-3p agomir), group B(group agomir-NC), group C(group model) and group D(group sham), with 10 rats in each group, respectively. The intrathecal injection of miR-124-3p agomir(group A), agomir-NC(group B) or saline(group C, D) was given to the spinal tissue of T10 using modified Sloane′s method at one day before the spinal cord injury modeling. The Allen′s method was used to establish the spinal cord injury model, and the group D was given surgical treatments except spinal cord injuring. Basso-Beai-Bresnehan scale (BBB)was scored prior to and one day after the spinal cord injury. The level of miR-124-3p in T10 spinal cord was evaluated by qRT-PCR before and 1 day, 3 days and 5 days after surgery. Neuronal apoptosis and expression ofcaspase 3, 68KD NFP proteins in spinal cord tissue were detected using TUNEL staining and western blot. Elisa kit was used to detect ROS, MDA, SOD and CAT. Targetscan and double luciferase reporter experiments were used to predict and validate the downstream target of miR-124-3p. The effect of target protein on neuronal apoptosis was detected using flow cytometry and western blot. Results: The BBB score in group A(12.57±1.42) was significantly higher than that in group B(6.31±1.03, P<0.05) and group C(6.18±1.15, P<0.05). The miR-124-3p expression in spinal tissue in group A(9.34±1.35) was also higher than that in group B(0.81±0.16, P<0.05) and group C(0.85±0.18, P<0.05). Upregulation of the miR-124-3p expression significantly decreased the number of apoptotic neurons(group A, 28.31±5.43; group B, 54.97±9.25; group C, 56.31±8.11; group D, 14.65±4.81) and activated caspase 3 level in spinal cord tissue (group A, 1.65±0.17; group B, 3.24±0.33; group C, 3.26±0.34; group D, 1.00±0.08) and significantly increased the level of 68KD NFP (group A, 0.69±0.08; group B, 0.25±0.04; group C, 0.23±0.03; group D, 1.00±0.09, P<0.05). In addition, upregulation of miR-124-3p can significantly reduce ROS(group A, 1.29±0.10; group B, 1.67±0.13; group C, 1.65±0.12, P＜0.05) and MDA levels (group A, 1.43±0.12; group B, 1.74±0.13; group C, 1.77±0.16, P＜0.05), and significantly increase SOD(group A, 0.72±0.08; group B, 0.34±0.05; group C, 0.36±0.06, P＜0.05) and CAT levels(group A, 0.51±0.07; group B, 0.26±0.03; group C, 0.27±0.04, P<0.05). Targetscan prediction showed that there was a sequence binding at the 3′UTR end of miR-124-3p and calpain 1. Compared to transfection of agomir-NC and calpain 1 wild sequences, the fluorescence intensity decreased significantly after transfection of the wild sequences of miR-124-3p agomir and calpain 1(site 91-97: 35.16±4.39 vs 101.25±9.98; site 467-473: 55.41±8.16 vs 98.84±10.45, P<0.05). After down-regulation of calpain 1 expression, the apoptotic rate and activated caspase 3 level of neurons significantly decreased (group shRNA-calpain1: 1.67±0.18; group shRNA-con: 2.52±0.27; group con: 1.00±0.12), while the 68KD NFP level increased significantly(group shRNA-calpain1: 0.71±0.06; group shRNA-con: 0.36±0.04; group con: 1.00±0.14, P<0.05). Conclusions: miR-124-3p inhibits neuronal apoptosis induced by oxidative stress and alleviates spinal cord injury by inhibiting calpain 1 expression through 3′UTR.