创伤性脑损伤激活miR-124-3p/Notch通路促进大鼠神经干细胞增殖和分化

Activation of miR-124-3p/Notch pathway promotes proliferation and differentiation of rat neural stem cells after traumatic brain injury

目的观察创伤性脑损伤(TBI)大鼠伤侧海马区微小RNA-124-3p(miR-124-3p)的表达变化,探讨miR-124-3p对大鼠TBI后神经再生的影响。方法将健康雄性大鼠按随机数字表法分为假手术组、 TBI组、 miR-124-3p激动剂(miR-124-3p agomir)组与miR-124-3p抑制剂(miR-124-3p antagomir)组,后3组大鼠应用控制性皮层撞击(CCI)法建立TBI模型。创伤后, miR-124-3p agomir组与miR-124-3p antagomir组通过由创伤对侧侧脑室分别注射miR-124-3p agomir或miR-124-3p antagomir各1 nmoL,假手术组和TBI组注入等量溶剂。造模后12 h、 1 d、 3 d、 7 d,实时定量PCR检测大鼠伤侧海马区组织miR-124-3p的表达, Western blot法检测Delta样分子1(DLL1)的表达水平,免疫荧光组织化学染色检测海马组织5-溴脱氧尿嘧啶核苷(BrdU)、神经元核抗原(NeuN)、神经上皮干细胞蛋白(nestin)的表达。生物信息学软件预测并通过荧光素酶报告实验验证miR-124-3p与DLL1之间的靶向结合作用。结果与假手术组相比, TBI组大鼠海马区miR-124-3p和DLL1的表达均显著升高;与TBI组比较, miR-124-3p agomir组miR-124-3p的表达量明显升高、 DLL1表达量明显减少, miR-124-3p antagomir组miR-124-3p表达量明显降低, DLL1表达量则明显升高。创伤后7 d, TBI大鼠海马组织BrdU^+NeuN^+细胞与BrdU^+nestin^+细胞数量明显多于假手术组, miR-124-3p agomir处理增加BrdU^+NeuN^+细胞与BrdU^+nestin^+细胞数量, miR-124-3p antagomir处理则减少BrdU^+NeuN^+细胞与BrdU^+nestin^+细胞数量。生物信息学分析证实DLL1为miR-124-3p的靶基因。结论创伤区miR-124-3p的高表达可靶向抑制DLL1蛋白表达,促进神经干细胞增殖和分化。

Objective To explore the change of the expression of microRNA-124-3p(miR-124-3p) in injured hippocampus of rats and investigate the role of miR-124-3p in neuranagenesis after traumatic brain injury(TBI). Methods The healthy male rats were randomly divided into a sham-operated group, TBI group, miR-124-3p agomir group and miR-124-3p antagomir group. TBI models were constructed by controlled cortical injury(CCI) device for all the groups except for the sham-operated group. The miR-124-3p agomir(1 nmol) was given to the miR-124-3p agomir group and miR-124-3p antagomir(1 nmol) to the miR-124-3p antagomir group via lateral ventricular injection, and equivalent solvent was given to the sham-operated group and TBI group after injury. The injured hippocampus of rats was collected at 12 hours, 1 day, 3, 7 days after injury. The real-time PCR and Western blot analysis were used to examine the expression of miR-124-3p and Delta-like 1(DLL1) in the injured hippocampus. Immunofluorescence histochemistry was used to examine the expression levels of 5-bromodeoxyuridine(BrdU), neuronal nuclear antigen(NeuN) and nestin in the injured hippocampus. Bioinformatics software was used to predict and dual luciferase reporter assay to validate the regulatory relationship between miR-124-3p and DLL1. Results The miR-124-3p and DLL1 expression in the TBI group were significantly higher than those in the sham-operated group;compared with the TBI group, the miR-124-3p agomir group had significantly increased expression of miR-124-3p and significantly decreased expression of DLL1 in the injured hippocampus, and miR-124-3p antagomir group had significantly decreased expression of miR-124-3p and significantly increased expression of DLL1. Compared with the sham-operated group, the BrdU^+NeuN^+ cells and BrdU^+nestin^+ cells in the hippocampus significantly increased in the TBI group at 7 days after injury. The miR-124-3p agomir treatment increased the number of the BrdU^+NeuN^+ cells and BrdU^+nestin^+ cells, while the miR-124-3p antagomir treatment decreased the number of the BrdU^+NeuN^+ cells and BrdU^+nestin^+ cells. Bioinformatics analysis confirmed that DLL1 was a target of miR-124-3p. Conclusion High expression of miR-124-3p in the trauma region promotes the proliferation and differentiation of neural stem cells by targeting and inhibiting DLL1.