Abstract：
Cerebral ischemia is a severe neurological disability related to neuronal apoptosis and cellular stress response. Circular RNAs (circRNAs) are emerging regulators of cerebral ischemia. Herein, this study proposed to probe the action of circ_0000115 in cerebral ischemia injury. The mouse neuroblastoma cells N2a and HT22 underwent oxygen-glucose deprivation (OGD) were used as a model of in vitro cerebral ischemia. Levels of genes and proteins were detected by qRT-PCR and western blotting. Cell proliferation and apoptosis were determined by EdU assay and flow cytometry. Western blotting was used to detect the protein level of pro-inflammatory factors. The oxidative stress injury was evaluated by detecting reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) generation. Dual-luciferase reporter and RIP assays were used to confirm the target relationship between miR-1224-5p and circ_0000115 or nitric oxide synthase 3 (NOS3). OGD exposure decreased circ_0000115 and NOS3 expression, and increased miR-1224-5p in N2a and HT22 cells in a time-dependent manner. Circ_0000115 silencing attenuated OGD-induced apoptosis, oxidative stress and inflammation in N2a and HT22 cells. Mechanistically, circ_0000115 directly sponged miR-1224-5p, which targeted NOS3. Furthermore, rescue experiments showed that miR-1224-5p overexpression abolished the neuroprotective effect of circ_0000115 in N2a and HT22 cells under OGD treatment. Besides that, silencing of miR-1224-5p protected N2a and HT22 cells against OGD-evoked injury, which was counteracted by NOS3 knockdown. Circ_0000115 protects N2a and HT22 cells against OGD-evoked neuronal apoptosis, inflammation, and oxidative stress via the miR-1224-5p/NOS3 axis, providing an exciting view of the pathogenesis of cerebral ischemia.
摘要：
脑缺血是一种与神经元凋亡和细胞应激反应有关的严重神经系统残疾。环状RNA（circularRNAs）是脑缺血的新兴调节因子。在这里，本研究旨在探讨circ_0000115在脑缺血损伤中的作用。小鼠神经母细胞瘤细胞N2a和HT22经历氧糖剥夺（OGD）作为体外脑缺血模型。通过qRT-PCR和蛋白质印迹检测基因和蛋白质的水平。用EdU法和流式细胞术测定细胞增殖和凋亡。采用免疫印迹法检测促炎因子的蛋白水平。通过检测活性氧（ROS）来评估氧化应激损伤，丙二醛（MDA）和超氧化物歧化酶（SOD）的生成。使用双荧光素酶报告基因和RIP测定来确认miR-1224-5p与circ_0000115或一氧化氮合酶3(NOS3)之间的靶关系。OGD暴露降低了circ_0000115和NOS3表达，N2a和HT22细胞中miR-1224-5p呈时间依赖性增加。Circ_0000115沉默减弱OGD诱导的细胞凋亡，N2a和HT22细胞的氧化应激和炎症反应。机械上,circ_0000115直接海绵化miR-1224-5p,针对NOS3。此外，拯救实验表明miR-1224-5p过表达在OGD处理下消除了circ_0000115在N2a和HT22细胞中的神经保护作用。除此之外,沉默miR-1224-5p保护N2a和HT22细胞免受OGD诱发的损伤，这被NOS3击倒所抵消。Circ_0000115保护N2a和HT22细胞免受OGD诱发的神经元凋亡，炎症,和氧化应激通过miR-1224-5p/NOS3轴，为脑缺血的发病机制提供了一个令人兴奋的观点。
