miR-124-3p靶向作用于calpain 1抑制氧化应激对脊髓损伤后神经元凋亡的影响

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

中文关键词:  脊髓损伤  miR-124-3p  钙蛋白酶1  神经元凋亡  氧化应激

英文关键词:Spinal cord injury  MiR-124-3p  Calpain 1  Apoptosis  Neurons

中文摘要:

  【摘要】 目的：探究microRNA（miR）-124-3p作用于calpain 1抑制氧化应激对脊髓损伤后神经元凋亡的影响及其分子机制。方法：选取成年雄性SD大鼠40只，随机分成A组（miR-124-3p agomir组）、B组（agomir-NC组）、C组（model组）以及D组（sham组），每组10只。构建脊髓损伤模型前1d，采用改良的Sloane法向大鼠的脊髓组织T10段蛛网膜下腔注射miR-124-3p agomir（A组）、agomir-NC（B组）或生理盐水（C组、D组）。Allen法建立脊髓损伤模型，D组行手术处理但不进行脊髓撞击。脊髓损伤术前及术后1d进行Basso-Beai-Bresnehan（BBB）量表评分，qRT-PCR检测术前及术后1d、3d、5d模型大鼠T10段脊髓中miR-124-3p水平。通过TUNEL染色和Western blot检测脊髓组织神经元细胞凋亡情况以及caspase 3、68KD 神经丝蛋白（neurofilament protein，NFP）的表达。利用Elisa试剂盒检测总活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）、过氧化氢酶（catalase，CAT）以及超氧化物歧化酶（superoxide dismutase，SOD）。利用Targetscan和双荧光素酶报告实验预测并验证miR-124-3p下游靶点。通过流式细胞术和Western blot检测靶点蛋白对神经元细胞凋亡的影响。结果：A组术后1d的BBB评分（12.57±1.42）明显高于B组（6.31±1.03，P＜0.05）和C组（6.18±1.15，P＜0.05），A组脊髓组织中miR-124-3p表达水平（9.34±1.35）高于B组（0.81±0.16，P＜0.05）和C组（0.85±0.18，P＜0.05）。上调miR-124-3p表达水平显著降低了脊髓组织中凋亡神经元细胞数量（A组，28.31±5.43；B组，54.97±9.25；C组，56.31±8.11；D组，14.65±4.81）和激活的caspase 3水平（A组，1.65±0.17；B组，3.24±0.33；C组，3.26±0.34；D组，1.00±0.08），同时显著上调了68KD NFP水平（A组，0.69±0.08；B组，0.25±0.04；C组，0.23±0.03；D组，1±0.09，P＜0.05）。进一步检测发现，上调miR-124-3p能够显著下调ROS（A组，1.29±0.10；B组，1.67±0.13；C组，1.65±0.12，P＜0.05）和MDA水平（A组，1.43±0.12；B组，1.74±0.13；C组，1.77±0.16，P＜0.05），明显提高SOD（A组，0.72±0.08；B组，0.34±0.05；C组，0.36±0.06，P＜0.05）和CAT水平（A组，0.51±0.07；B组，0.26±0.03；C组，0.27±0.04，P＜0.05）。Targetscan预测发现miR-124-3p和calpain 1 mRNA的3′UTR端存在序列结合。转染miR-124-3p agomir和calpain 1野生序列后，与转染agomir-NC和calpain 1野生序列相比，荧光强度显著降低（91-97位点：35.16±4.39 vs 101.25±9.98；467-473位点：55.41±8.16 vs 98.84±10.45，P＜0.05）。下调calpain 1表达后，神经元细胞凋亡率和激活的caspase 3水平明显降低（shRNA-calpain1组，1.67±0.18；shRNA-con组，2.52±0.27；con组，1.00±0.12），而 68KD NFP水平显著升高（shRNA-calpain1组，0.71±0.06；shRNA-con组，0.36±0.04；con组，1.00±0.14，P＜0.05）。结论：miR-124-3p通过3′UTR抑制calpain 1表达，从而抑制氧化应激导致的神经元凋亡，减轻脊髓损伤。

英文摘要:

  【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.