miR-25调控Notch信号通路对中枢神经系统感染小鼠记忆容量和脑损伤的影响  

Effect of the miR-25-mediated Notch signaling pathway on memory capacity and brain injury of mice with central Nervous system infection

作　　者：李成 黄学卿 王黎洲[1] 周石[1] 许敏[1] 安天志[1] LI Cheng;HUANG Xueqing;WANG Lizhou;ZHOU Shi;XU Min;AN Tianzhi(School of Medical Imaging,the Affiliated hospital of guizhou medical university,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学影像学院,贵州贵阳550004 [2]贵州医科大学

出　　处：《贵州医科大学学报》2023年第1期17-24,共8页Journal of Guizhou Medical University

基　　金：国家自然科学基金(82060333)。

摘　　要：目的探讨miR-25调控Notch信号通路对中枢神经系统(CNS)感染小鼠记忆容量和脑损伤的影响。方法采用生物学预测网站(www.targetscan.org)分析微小RNA-25(miR-25)和Notch1的结合位点,将1个Rellina质粒和2个报告质粒分别与miR-25质粒、空载体质粒共同转染入人类胚肾细胞HEK293T细胞,采用双萤光素酶报告基因系统验证miR-25和Notch1的关联;70只健康雄性C57BL/6小鼠分为正常对照组(不作处理,n=10)和实验组(建立CNS感染模型,n=60),实验组小鼠造模成功后随机均分为模型组(无处理)、空载体组(注射空载体)、miR-25模拟物组(注射miR-25模拟物)、miR-25抑制剂组(注射miR-25抑制剂)、(2S)-N-[N-(3,5-二氟苯乙酰基)-L-丙氨酰]-2-苯基甘氨酸叔丁酯(DAPT)组(注射DAPT)及miR-25抑制剂+DAPT组(注射miR-25抑制剂+DAPT);采用莫里斯水迷宫检测各组小鼠的学习、记忆容量(逃生潜伏期、平台停留时间及经过次数);麻醉处死各组小鼠后取脑组织,采用实时荧光定量PCR(qRT-PCR)检测各组小鼠大脑海马CA1区miR-25、Notch1及Hes家族BHLH转录因子5(Hes5)的表达,采用Western blot检测各组小鼠大脑海马CA1区Notch1、Hes5、环加氧酶2(COX-2)及诱导型一氧化氮合酶(iNOS)的表达,采用水溶性四氮唑(WST-1)法和硫代巴比妥酸实验检测各组小鼠海马组织中的超氧化物歧化酶(SOD)和丙二醛(MDA)水平。结果miR-25与Notch1的3′UTR结合序列为UGCAAU,双萤光素酶报告基因结果提示miR-25可以负调控Notch1的表达;和正常对照组比较,实验组小鼠大脑海马CA1区miR-25、COX-2及iNOS的表达升高,Notch1和Hes5表达下降,海马组织中SOD水平降低、MDA水平升高,差异均有统计学意义(P<0.05);miR-25模拟物组和DAPT组小鼠大脑海马组织中Notch1和Hes5表达下降,学习、记忆容量降低,海马组织中SOD水平降低、MDA水平升高,COX-2和iNOS表达上升,差异均有统计学意义(P<0.05)。结论miR-25下调可靶向激活调控CNS感染小�Objective To explore the effect of miR-25 on memory capacity and brain injury in mice with central nervous system(CNS)infection via the Notch signaling pathway.Methods The binding sites between microRNA-25(miR-25)and Notch1 were predicted using a bioinformatics website(www.targetscan.org).HEK293 T cells were co-transfected with a reporter vector carrying Rellina luciferase,a luciferase reporter carrying miR-25 or a luciferase reporter carrying empty(empty vector).The binding of miR-25 to Notch1 was verified using a dual-luciferase reporter gene assay.Seventy healthy male C57 BL/6 mice were divided into normal control group(no treatment,n=10)and experimental group(established CNS infection model,n=60).After successful modeling,the mice were randomly divided into the following groups according to injected agents:model(no further treatment),empty vector,miR-25 mimics,miR-25 inhibitor,(2 S)-N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-2-phenylglycine tert butyl ester(DAPT)and miR-25 inhibitor+DAPT.Morris water maze experiment was performed to measure the learning and memory capacity(escape latency,the time spent on the platform and platform crossover number)of mice in each group.After the mice in each group were anesthetized and sacrificed,the brain tissues were taken.Quantitative real-time polymerase chain reaction(qRT-PCR)was performed to measure the expression of miR-25,Notch1 and HES family bHLH transcription factor 5(Hes5)in the mouse hippocampal CA1 region.Western blot was used to detect the expression of Notch1,Hes5,cyclooxygenase 2(COX-2)and inducible nitric oxide synthase(iNOS)in mouse hippocampal CA1 region.The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in mouse hippocampus in each group were detected by water-soluble tetrazolium assay and thiobarbituric acid test,respectively.Results 3’UTR binding sequence of Notch1 to miR-25 was UGCAAU.Dual luciferase reporter assay results suggested that miR-25 negatively regulated Notch1 expression.When compared with the normal control group,miR-25,COX-2

关 键 词：中枢神经系统感染 脑损伤 微小RNA-25 记忆容量 NOTCH信号通路 环加氧酶2 诱导型一氧化氮合酶 

分 类 号：R522[医药卫生—内科学] Q426[医药卫生—临床医学]