MicroRNA-124通过调控PTEN促进脑缺血损伤小鼠神经轴突生长和运动功能恢复

MicroRNA-124 promotes neurite elongation and motor function recovery in mice with cerebral ischemia injury by targeting PTEN

目的探究MicroRNA-124(miR-124)对脑缺血损伤小鼠神经功能的改善作用及其内在机制。方法①取32只C57小鼠随机分为假手术组和脑缺血组(n=16),TTC染色和神经行为学评分检测小鼠脑损伤后3天脑梗死面积和运动情况,RT-qPCR检测miR-124在两组间的表达变化。②培养原代神经元细胞,通过氧糖剥夺模拟缺血再灌注损伤,观察氧糖剥夺后0、72 h miR-124的变化趋势。③另取32只C57小鼠随机分为脑缺血组和脑缺血+miR-124组(n=16),TTC染色、转棒实验和肢体放置实验检测小鼠的运动功能改善情况。④细胞实验分为氧糖剥夺组和氧糖剥夺+miR-124组,通过免疫荧光实验检测两组神经元轴突的生长情况。Targetscan数据库预测miR-124下游靶基因,双荧光素酶基因报告和Western blot在原代神经元等上验证对照组和miR-124组下游靶基因第10号染色体缺失的磷酸酶及张力蛋白同源基因(the phosphate and tensin homolog deleted on chromosome ten,PTEN)的表达变化。上调PTEN,检测氧糖剥夺+miR-124和氧糖剥夺+miR-124+PTEN两组神经元轴突的生长情况。(5)在体实验中检测脑缺血+miR-124组和脑缺血+miR-124+Pten组(n=8)转棒实验和肢体放置情况。结果脑缺血再灌注后,小鼠脑梗死面积约达到40%(P<0.001),小鼠的神经认知功能显著降低(P<0.001),已达到重度认知功能损害;miR-124的表达较对照组明显升高(P<0.05);而进一步过表达miR-124能够促进氧糖剥夺后神经元轴突的生长(P<0.05),显著缩小脑梗死面积(P<0.05),且促进动物运动能力的恢复和改善(P<0.05);Targetscan数据库预测miR-124下游靶基因为PTEN,双荧光素酶基因报告和Western blot都证实miR-124能有效下调PTEN的表达(P<0.05);最后,上调PTEN逆转了miR-124促进神经元轴突的生长以及改善小鼠运动能力的效应(P<0.05)。结论miR-124通过下调PTEN促进脑缺血小鼠神经元轴突生长,且有助于小鼠运动功能的恢复。

Objective To explore the effect of microRNA-124(miR-124)on the improvement of neurological function in mice with cerebral ischemia injury and its underlying mechanism.Methods①A total of 32 C57 mice were randomly divided into sham group and cerebral ischemia group(n=16).The cerebral infarction area was detected by TTC staining and motor status of the mice was evaluated with neurobehavioral scoring in 3 d after brain injury.The expression change of miR-124 was detected by RT-qPCR and compared between the 2 groups.②Primary neurons were isolated from fetal mice and cultured,and ischemia-reperfusion injury was simulated by oxygen glucose deprivation.The changes of miR-124 at 0 and 72 h after oxygen glucose deprivation were observed.③Another 32 C57 mice were randomly divided into cerebral ischemia group and cerebral ischemia+miR-124 group(n=16).TTC staining,rotating rod test and limb placement test were performed respectively to detect whether the motor function of the mice was improved.④The primary neurons were divided into oxygen-glucose deprivation group and oxygen-glucose deprivation+miR-124 group.The neurite elongation in the 2 groups was detected by immunofluorescence assay.The downstream target gene of miR-124 was predicted with Targetscan database.Double luciferase gene reporter assay and Western blotting were used to verify the effectiveness of miR-124 regulation on phosphatase and tensin homolog deleted on chromosome 10(PTEN)in the control and miR-124 groups.Then,the effect of PTEN up-regulation on neurite elongation was observed in the oxygen glucose deprivation+miR-124 cells and oxygen glucose deprivation+miR-124+PTEN groups.(5)Rotating rod test and limb placement test were performed again for motor functions in the mice of cerebral ischemia+miR-124 group and cerebral ischemia+miR-124+PTEN group(n=8).Results Cerebral ischemia-reperfusion resulted in the area of cerebral infarction about 40%(P<0.001)and significantly reduced neurocognitive function in mice(P<0.001),reaching the condition of severe cognitive impairment.The expression of miR-124 was significantly higher in the cerebral ischemia group than the control group(P<0.05).Overexpression of miR-124 could promote the growth of neuronal axons after oxygen glucose deprivation(P<0.05),reduce the area of cerebral infarction(P<0.05),and thus promote the recovery and improvement of animal motor ability(P<0.05).With the aid of Targetscan database,PTEN was predicted as the downstream target of miR-124,and double luciferase gene reporter assay and Western blotting confirmed that miR-124 could effectively down-regulate the expression of PTEN(P<0.05);Finally,up-regulation of PTEN reversed the effect of miR-124 on promoting the growth of neuronal axons and improving the motor ability of mice(P<0.05).Conclusion MiR-124 promotes neuronal axonal growth and contributes to the recovery of motor function in mice with cerebral ischemia by down-regulating PTEN.