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

作　　者：任翔 靖颖霞[2] 周芝文[1] Ren Xiang;Jing Ying-Xia;Zhou Zhi-Wen(Department of Neurology,Hunan Provincial People's Hospital(The First Affiliated Hospital of Hunan Normal University),Changsha,Hunan 410016,China;Department of Emergency,Hunan Provincial People's Hospital(The First Affiliated Hospital of Hunan Normal University),Changsha,Hunan 410016,China)

机构地区：[1]湖南省人民医院(湖南师范大学附属第一医院)神经内科,湖南长沙410016 [2]湖南省人民医院(湖南师范大学附属第一医院)急诊科,湖南长沙410016

出　　处：《中国现代医学杂志》2024年第22期32-42,共11页China Journal of Modern Medicine

基　　金：湖南省卫生健康委科研计划项目(No:202103071026)。

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、pAkt/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。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(qRTPCR).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,pPI3K,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-1βand 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-1βand 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

关 键 词：缺血性脑卒中 miR-17-5p Spry1 氧化应激 

分 类 号：R743.3[医药卫生—神经病学与精神病学]