下调miR-205抑制皮质神经元的缺血性损伤

Down-regulation of microRNA-205 suppresses the ischemic injury of cortical neurons

目的:探讨miR-205对皮质神经元缺氧/缺糖损伤的作用与其可能的作用机制。方法:大鼠大脑皮层神经细胞的分离培养和MAP2免疫荧光鉴定皮质神经元。缺氧/缺糖处理构建皮质神经元缺血性损伤模型。实时荧光定量PCR检测miR-205表达量。利用Lipofectamine2000转染miR-205 mimics,miR-205抑制剂或LRP-1 siRNA。试剂盒检测caspase-3活性。Annexin-V fluorescein isothiocyanate conjugate and propidium iodide(Annexin-V FITC/PI)方法检测细胞凋亡。MTT实验检测细胞生长活力。双荧光素酶报告基因检测miR-205与LRP-1的靶向调控关系。Western Blot检测LRP-1蛋白表达量。结果:MAP2鉴定为皮质神经元。皮质神经元缺氧/缺糖损伤下调miR-205表达量。细胞转染实验中,下调miR-205显著抑制损伤模型的细胞凋亡和caspase-3活性,提高细胞生长活力;过表达miR-205则作用相反。对于miR-205的靶向基因LRP-1,过表达miR-205可显著抑制LRP-1,而抑制miR-205其作用则相反。同时抑制miR-205和LRP-1促进损伤模型的细胞凋亡和caspase-3活性,降低细胞生长活力。结论:下调miR-205可以通过调控LRP-1抑制皮质神经元的缺血性损伤,为新生儿脑卒中的预防和治疗提供一定的理论基础。

Objective： To investigate the effect of miR-205 on cerebral cortical neuron with anoxia/hypoglycemia injury and the possible related mechanism. Methods： The rat cerebral cortical neurons were isolated and characterized by the MAP2 immunofluorescence. The cellular model of cerebral ischemia model was established by culturing under anoxia/hypoglycemie condition. The expression of miR-205 was measured by quantitative real-time PCR. The transfection of miR-205 mimics, miR-205 inhibitors and LRP-1 siRNA using Lipofectamine2000 s. The activity of Caspase-3 was de- tected using a commercial kit . Annexin-V fluorescein isothioeyanate conjugate and propidium iodide（ Annexin-V FITC/ PI） was used to measure cells apoptosis by flow cytometry. MTr assay is used for detection of cellular viability. The targeted relationship between miR-205 and LRP-1 was detected using dual luciferase reporter assay. Protein expression of LRP1 was analyzed by Western Blotting. Results： The cortical neuron was identified by MAP2 immunofluoreseence. miR-205 was down-regulated by anoxia/hypoglycemic injury in cortical neuron, miR-205 inhibition significantly suppressed Caspase-3 activity and cells apoptosis while promoting cells viability in cortical neuron with anoxia/hypoglycemic injury, but miR-205 overexpression had the reverse effects. LRP-1 is a direct target of miR-205, and overexpression of miR-205 suppressed the expression of LRP-1 while miR-205 inhibition had the converse effect. The co-inhibition of miR-205 and LRP-1 increased the cells apoptosis and Caspase-3 activation, while decreased cells viability in cortical neuron with anoxia/hypoglycemic injury. Conclusion： Down-regulation of miR-205 suppresses cortical neuronal ischemic injury via upregulating LRP-1, providing a theoretical reference base for the prevention and treatment of neonatal stroke.