MicroRNA-17-5p调控Spry1缓解脑缺血再灌注损伤的机制研究

Mechanism by which microRNA-17-5p regulates Spry1 to alleviate cerebral ischemia-reperfusion injury

摘要:

目的 探究miR-17-5p通过靶向Spry1调控PI3K/Akt介导氧糖剥夺/复氧（OGD/R）细胞模型氧化应激及细胞增殖凋亡的分子机制，以阐明脑缺氧损伤的发病机制。方法 通过OGD/R处理人脑微血管内皮细胞构建细胞模型，通过实时荧光定量聚合酶链反应（qRT-PCR）检测miR-17-5p和Spry1 mRNA表达；采用CCK-8法、流式细胞术检测细胞增殖、细胞凋亡；通过生物信息学网站StarBase预测miR-17-5p和Spry1的互补结合位点，并通过双萤光素酶实验分析两者的结合关系；Western blotting检测Spry1、p-PI3K、PI3K、p-Akt、Akt蛋白相对表达量；试剂盒检测细胞中活性氧（ROS）、超氧化物歧化酶（SOD）和丙二醛（MDA）水平；酶联免疫吸附试验测定肿瘤坏死因子α（TNF-α）、白细胞介素-1β（IL-1β）和IL-6浓度。结果 与对照组相比，OGD/R处理导致miR-17-5p表达降低（P <0.05），Spry1水平升高（P <0.05），细胞活力降低（P <0.05），细胞凋亡率增加（P <0.05），ROS相对荧光强度和MDA水平升高，SOD水平降低（P <0.05），TNF-α、IL-1β和IL-6升高（P <0.05），p-PI3K/PI3K、p-Akt/Akt蛋白水平降低（P <0.05）；与OGD/R组相比转染miR-17-5p mimics或者Spry1敲降载体后，细胞活力升高（P <0.05），细胞凋亡率减少（P <0.05），ROS相对荧光强度和MDA水平降低，SOD水平升高（P <0.05），TNF-α、IL-1β和IL-6降低（P <0.05），p-PI3K/PI3K、p-Akt/Akt蛋白水平升高（P <0.05）；双萤光素酶实验证实miR-17-5p和Spry1存在互补结合；同时转染miR-17-5p mimics和Spry1过表达载体后与OGD/R组相比，各指标均差异无统计学意义（P >0.05）。结论 miR-17-5p通过靶向Spry1调控PI3K/Akt介导OGD/R细胞模型氧化应激及细胞的增殖和凋亡。

Abstract:

Objective To explore the molecular mechanism by which miR-17-5p targeted Spry1 to regulate the PI3K/Akt pathway, mediating oxidative stress, cell proliferation, and apoptosis in an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model, and so as to elucidate the pathogenesis of cerebral hypoxic injury.Methods Human brain microvascular endothelial cells were treated with OGD/R to construct a cell model. The expression levels of miR-17-5p and Spry1 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The CCK-8 assay and flow cytometry were used to detect the levels of cell proliferation and apoptosis. The binding sites between miR-17-5p and Spry1 were predicted using the bioinformatics website StarBase, and the binding relationship between them was analyzed by a dual-luciferase reporter assay. The protein levels of Spry1, p-PI3K, PI3K, p-Akt and Akt were detected by Western blotting. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) in cells were detected with respective kits. The concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 were measured using an enzyme-linked immunosorbent assay (ELISA) .Results Compared with the control group, OGD/R treatment led to decreased expression of miR-17-5p (P < 0.05), increased levels of Spry1 (P < 0.05), lower cell viability (P < 0.05), higher apoptosis rates (P < 0.05), increased levels of ROS and MDA but decreased levels of SOD (P < 0.05), increased levels of TNF-α, IL-1β and IL-6 (P < 0.05), and decreased p-PI3K/PI3K and p-Akt/Akt protein levels (P < 0.05). Compared with the OGD/R group, transfection with miR-17-5p mimics or Spry1 knockdown vectors enhanced the cell viability (P < 0.05), reduced the apoptosis rate (P < 0.05), lowered the levels of ROS and MDA but elevated the levels of SOD (P < 0.05), downregulated the concentrations of TNF-α, IL-1β and IL-6 (P < 0.05), and upregulated the p-PI3K/PI3K and p-Akt/Akt protein levels (P < 0.05). The dual-luciferase reporter assay confirmed the binding between miR-17-5p and Spry1. After co-transfection with miR-17-5p mimics and the Spry1 overexpression vectors, there was no significant difference in any indicators compared with the OGD/R group (P > 0.05).Conclusions MiR-17-5p regulates PI3K/Akt by targeting Spry1 to mediate oxidative stress, cell proliferation and apoptosis in the OGD/R cell model.