Abstract：
Stroke is an acute cerebrovascular disease that is now the most important cause of death due to brain problems in our country. CircRNAs are RNA circles that have been extensively involved in the disease. We aimed to investigate the mechanism of circ_0129657 in the pathogenesis of stroke. In this study, quantitative real-time polymerase chain reaction (RT-qPCR) and western blot assays were used to assess the expression of circ_0129657, miR-194-5p, and glia maturation factor beta (GMFB). Cell viability was measured by Cell Counting Kit-8 (CCK-8) assay. 5-Ethynyl-2\'-Deoxyuridine (EdU) assay was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays were used to assess the relationship between miR-194-5p and circ_0129657 or GMFB. Mouse middle cerebral artery occlusion (MCAO) model was applied to mimic the cerebral ischemia/reperfusion injury. Our data showed that the levels of circ_0129657 and GMFB were significantly increased and the expression of miR-194-5p was significantly decreased in oxygen-glucose deprivation (OGD)-induced human brain microvascular endothelial cells (HBMECs). Silencing circ_0129657 expression in OGD-induced HBMECs could promote cell viability and cell proliferation. Moreover, circ_0129657 depletion also could inhibit apoptosis and inflammatory factor secretion. Circ_0129657 functioned as a sponge for miR-194-5p and could regulate GMFB expression via miR-194-5p competition. Furthermore, miR-194-5p downregulation or GMFB restoration could partially reverse the effects of circ_0129657 silencing on cell biological properties in OGD-induced HBMECs. Meanwhile, circ_0129657 knockdown decreased cerebral infarction volume and neurological impairment in MCAO mouse models. In conclusion, our findings suggest that circ_0129657 can inhibit cell proliferation and promote apoptosis and inflammatory factor secretion in HBMECs after oxygen-glucose deprivation via miR-194-5p/GMFB axis, providing evidence that circ_0129657 has the potential as a useful biological molecular marker in the diagnosis of stroke.
摘要：
中风是一种急性脑血管病，目前是我国因脑部问题导致死亡的最重要原因。CircRNAs是广泛参与该疾病的RNA环。我们旨在探讨circ_0129657在中风发病中的作用机制。在这项研究中,定量实时聚合酶链反应（RT-qPCR）和蛋白质印迹测定用于评估circ_0129657，miR-194-5p，和胶质细胞成熟因子β(GMFB)。通过细胞计数试剂盒-8(CCK-8)测定测量细胞活力。5-乙炔基-2'-脱氧尿苷(EdU)测定法用于检测细胞增殖。流式细胞术检测细胞凋亡。双荧光素酶报告基因,RNA下拉，和RNA免疫沉淀(RIP)测定用于评估miR-194-5p与circ_0129657或GMFB之间的关系。应用小鼠大脑中动脉闭塞(MCAO)模子模仿脑缺血再灌注毁伤。我们的数据显示，在氧糖剥夺（OGD）诱导的人脑微血管内皮细胞（HBMECs）中，circ_0129657和GMFB的水平显着升高，miR-194-5p的表达显着降低。在OGD诱导的HBMECs中沉默circ_0129657表达可以促进细胞活力和细胞增殖。此外，circ_0129657耗竭还可以抑制细胞凋亡和炎症因子的分泌。Circ_0129657充当miR-194-5p的海绵,并且可以通过miR-194-5p竞争调节GMFB表达。此外，miR-194-5p下调或GMFB恢复可以部分逆转circ_0129657沉默对OGD诱导的HBMECs细胞生物学特性的影响。同时,circ_0129657敲除降低MCAO小鼠模型的脑梗死体积和神经功能缺损。总之,我们的发现表明circ_0129657可以通过miR-194-5p/GMFB轴抑制氧糖剥夺后HBMECs的细胞增殖，促进凋亡和炎症因子的分泌，提供证据表明circ_0129657具有作为中风诊断有用的生物分子标志物的潜力。
