长链非编码RNA MALAT1对氧糖剥夺/复氧诱导的人脑微血管内皮细胞血管生成的影响  

Effect of long non-coding RNA MALAT1 on angiogenesis of human brain microvascular endothelial cells induced by oxygenglucose deprivation/reoxygenation hypoxic

作　　者：高昶[1,2] 刘燕 杨皓翔 张翠翠 GAO Chang;LIU Yan;YANG Haoxiang;ZHANG Cuicui(Key Laboratory of Behavioral Medicine,School of Mental Health,Jining Medical University,Jining 272067,China;Graduate School,Jining Medical University,Jining 272067,China;Institute of Psychiatric Medicine,School of Mental Health,Jining Medical University,Jining 272013,China;Department of Neurology,Affiliated Hospital,Jining Medical University,Jining 272029,China;Department of Critical Care Medicine,Affiliated Hospital,Jining Medical University,Jining 272029,China)

机构地区：[1]济宁医学院精神卫生学院行为医学重点实验室,山东济宁272067 [2]济宁医学院研究生处,山东济宁272067 [3]济宁医学院精神卫生学院精神医学研究院,山东济宁272013 [4]济宁医学院附属医院神经内科,山东济宁272029 [5]济宁医学院附属医院重症医学三科,山东济宁272029

出　　处：《吉林大学学报（医学版）》2023年第4期832-839,共8页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(81901391);山东省政府泰山学者青年专家人才计划项目(tsqn201909145);山东省济宁市科技局重点研发计划项目(2019SMNS001);济宁医学院贺林基金项目(JYHL2021 MS04)。

摘　　要：目的:探讨长链非编码RNA (lncRNA)肺腺癌转移相关转录本1 (MALAT1)对氧糖剥夺/复氧(OGD/R)诱导的人脑微血管内皮细胞(HBMECs)血管生成的影响,并阐明其潜在的分子机制。方法:采用生物信息学方法预测MALAT1、不均一核糖核蛋白K (hnRNPK)和血管内皮生长因子A (VEGFA)的结合位点。HBMECs进行OGD/R处理构建脑缺血细胞模型,分为对照组、OGD/R模型组、OGD/R+siNC组、OGD/R+沉默MALAT1 (OGD/R+siMALAT1)组和OGD/R+siMALAT1+过表达VEGFA (OGD/R+siMALAT1+VEGFA)组。采用小干扰RNA (siRNA)沉默MALAT1的表达,采用pcDNA载体构建VEGFA过表达载体,将构建的siMALAT1和pcDNA VEGFA载体分别或同时转染至HBMECs中。利用管形成实验检测各组细胞血管形成能力,Western blotting法检测各组细胞中VEGFA蛋白表达水平。6周龄健康雄性C57BL/6 J小鼠20只,随机分为假手术组、大脑中动脉闭塞(MCAO)模型组、MCAO+NC空载体(MCAO+NC)组和MCAO+过表达MALAT1 (MCAO+MATAL1)组,每组5只,除假手术组外,其余各组小鼠利用线栓法构建MCAO小鼠模型,MCAO+NC组和MCAO+MATAL1组小鼠右脑室内分别注射NC空载体和MALAT1过表达载体。采用2,3,5-氯化三苯基四氮唑(TTC)染色检测各组小鼠脑梗死面积百分率,Western blotting法检测各组小鼠脑组织中VEGFA蛋白表达水平。结果:生物信息学预测,MALAT1与hnRNPK及hnRNPK与VEGFA均存在结合位点。管形成实验和Western blotting法检测,与对照组比较,OGD/R模型组细胞成管数明显增加(P<0.01),细胞中VEGFA蛋白表达水平明显升高(P<0.01);与OGD/R模型组比较,OGD/R+siMALAT1组细胞成管数明显减少(P<0.01),细胞中VEGFA蛋白表达水平明显降低(P<0.01);与OGD/R+siMALAT1组比较,OGD/R+siMALAT1+VEGFA组细胞成管数明显增加(P<0.01),细胞中VEGFA蛋白表达水平明显升高(P<0.01)。在MCAO模型小鼠实验中,TTC染色和Western blotting法检测,与假手术组比较,MCAO模型组小鼠脑梗死面积百分率和脑组织中VEGFA蛋白表达Objective:To discuss the effect of long non-coding RNA(lncRNA) lung cancer metastasisassociated transcript 1(MALAT1) on the angiogenesis of the human brain microvascular endothelial cells(HBMECs) induced by oxygen-glucose deprivatioin/reoxygenation(OGD/R),and to clarify its potential molecular mechanism.Methods:The binding sites of MALAT1,heterogeneous nuclear ribonucleoprotein K(hnRNPK),and vascular endothelial growth factor A(VEGFA) were predicted by bioinformatics.The HBMECs were treated with OGD/R to establish the cerebral ischemia cell model.The HBMECs were divided into control group,OGD/R model group,OGD/R+siNC group,OGD/R+ silencing MALAT1(OGD/R+siMALAT1) group and OGD/R+siMALAT1+over-expression of VEGFA(OGD/R+siMALAT1+VEGFA) group.Small interference RNA(siRNA) was used to silence the expression of MALAT1,and pcDNA vector was used to construct the VEGFA over-expression vector.The constructed siMALAT1 and pcDNA VEGFA vectos were transfected into the HBMECs separately or simultaneously.The angiogenesis abilities of cells in various groups were detected by tube formation assay;the expression levels of VEGFA protein in the cells in various groups were detected by Western blotting method.Twenty 6-week-old healthy male C57BL/6 J mice were randomly divided into sham operation group,middle cerebral artery occlusion(MCAO) model group,MCAO+NC blank vector(MCAO+NC) group,and MCAO+ over-expression of MALAT1(MCAO+MATAL1) group,and there were 5 mice in each group.Except for sham operation group,the mice in the other groups were used to construct the MCAO mouse models by suture method,and the NC empty vector and MALAT1 over-expression vector were injected into the right ventricle of the mice in MCAO+NC group and MCAO+MATAL1 group,respectively.The percentage of cerebral infarction area of mice in various groups were detected by 2,3,5-triphenyltetrazole chloride(TTC) staining,and the expression levels of VEGFA protein in brain tissue of mice in various groups were detected by Western blotting method.Results:The bioinformat

关 键 词：长链非编码RNA 肺腺癌转移相关转录本1 血管生成 不均一核糖核蛋白K 血管内皮生长因子A 

分 类 号：R741[医药卫生—神经病学与精神病学]