lncRNA MALAT1靶向miR-532-3p对脑出血继发脑损伤大鼠脑微血管内皮细胞模型凋亡和氧化损伤的影响  

Effect of lncRNA MALAT1 targeting miR-532-3p on apoptosis and oxidative damage of cerebral microvascular endothelial cell model in rats with secondary brain injury after intracerebral hemorrhage

作　　者：权瑜[1] 王举波[1] 程格庆 李娜[1] 李小冬 屈佩 王茂德[2] QUAN Yu;WANG Jubo;CHENG Geqing;LI Na;LI Xiaodong;QU Pei;WANG Maode(Department of Neurosurgery,Second Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710004,China;Department of Neurosurgery,First Affiliated Hospital of Xi’an Jiaotong University)

机构地区：[1]西安交通大学第二附属医院神经外科,西安710004 [2]西安交通大学第一附属医院神经外科

出　　处：《山西医科大学学报》2021年第7期874-882,共9页Journal of Shanxi Medical University

基　　金：陕西省自然科学基础研究计划项目(2017JC2-09)。

摘　　要：目的探讨长链非编码RNA(lncRNA)肺癌转移相关转录本1(MALAT1)靶向miR-532-3p对脑出血(ICH)继发脑损伤的大鼠脑微血管内皮细胞(RCMECs)凋亡和氧化损伤的影响。方法采用双重荧光素酶报告基因实验和实时定量PCR(RT-qPCR)确定MALAT1和miR-532-3p的靶向调控关系。首先将细胞分为对照组(正常培养的RCMECs)与模型(H_(2)O_(2))组(RCMECs氧化损伤模型),采用RT-qPCR分析并对比两组MALAT1和miR-532-3p的表达量。随后将细胞分为H_(2)O_(2)-si-NC组、H_(2)O_(2)-si-MALAT1组、H_(2)O_(2)-miR-mimics-NC组、H_(2)O_(2)-miR-532-3p mimics组、H_(2)O_(2)-si-MALAT1+miR-inhibitor-NC组、H_(2)O_(2)-si-MALAT1+miR-532-3p inhibitor组,分别转染si-NC、si-MALAT1、miR-mimics-NC、miR-532-3p mimics、共转染si-MALAT1与miR-inhibitor-NC、共转染si-MALAT1与miR-532-3p inhibitor后,构建RCMECs氧化损伤模型,然后采用流式细胞术分析RCMECs凋亡情况,采用试剂盒测定丙二醛(MDA)含量以及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,采用蛋白质印迹法(Wes-tern blot)检测B细胞淋巴瘤/白血病(Bcl)-2和Bcl相关x蛋白(Bax)表达量,并比较组间差异。结果MALAT1可与miR-532-3p特异性结合并负调控miR-532-3p表达(P<0.05)。与对照组比较,H_(2)O_(2)组中MALAT1的表达量显著升高,miR-532-3p的表达量显著降低,差异均有统计学意义(P<0.05)。与H_(2)O_(2)-si-NC组比,H_(2)O_(2)-si-MALAT1组沉默MALAT1可显著减弱H_(2)O_(2)诱导的RCMECs凋亡,并降低MDA含量、升高SOD和CAT活性、降低Bax蛋白相对表达量、升高Bcl-2蛋白相对表达量,差异均有统计学意义(P<0.05)。与H_(2)O_(2)-miR-mimics-NC组比,H_(2)O_(2)-miR-532-3p mimics组过表达miR-532-3p可显著减弱H_(2)O_(2)诱导的RCMECs凋亡,并降低MDA含量、升高SOD和CAT活性、降低Bax蛋白相对表达量、升高Bcl-2蛋白相对表达量,差异均有统计学意义(P<0.05)。与H_(2)O_(2)-si-MALAT1+miR-inhibitor-NC组比,H_(2)O_(2)-si-MALAT1+miR-532-3p inhibitor组沉默MALATObjective To investigate the effects of long non-coding RNA(lncRNA)lung cancer metastasis-related transcript 1(MALAT1)targeting miR-532-3p on apoptosis and oxidative damage of rat cerebral microvascular endothelial cells(RCMECs)after brain injury secondary to intracerebral hemorrhage(ICH).Methods Double luciferase reporter gene assay and real-time quantitative PCR(RT-qPCR)were used to determine the targeted regulatory relationship between MALAT1 and miR-532-3p.Firstly,cells were divided into control group(normal cultured RCMECs)and RCMEC oxidative damage model group(H_(2)O_(2)group),and RT-qPCR was used to analyze and compare the expression levels of MALAT1 and miR-532-3p between the two groups.Then cells were divided into H_(2)O_(2)-si-NC group,H_(2)O_(2)-si-MALAT1 group,H_(2)O_(2)-miR-mimics-NC group,H_(2)O_(2)-miR-532-3p mimics group,H_(2)O_(2)-si-MALAT1+miR-inhibitor-NC group and H_(2)O_(2)-si-MALAT1+miR-532-3p inhibitor group.After the cells were transfected with si-NC,si-MALAT1,miR-mimics-NC,miR-532-3p mimics,si-MALAT1 combined with miR-inhibitor-NC,si-MALAT1 combined with miR-532-3p inhibitor in six groups,respectively,RCMEC oxidative damage model was built.And then the apoptosis of RCMECs was analyzed by flow cytometry,the content of malondialdehyde(MDA)and the activities of superoxide dismutase(SOD)and catalase(CAT)were determined by kits,the expression levels of B-cell lymphoma/leukemia-2(Bcl-2)and Bcl-2-associated X(Bax)protein were detected by Western blot.Results MALAT1 could specifically bind to miR-532-3p and negatively regulated the expression of miR-532-3p(P<0.05).Compared with control group,the expression of MALAT1 in H_(2)O_(2)group was significantly increased,while the expression of miR-532-3p was significantly decreased(all P<0.05).Compared with H_(2)O_(2)-si-NC group,silencing MALAT1 in H_(2)O_(2)-si-MALAT1 group significantly reduced H_(2)O_(2)-induced RCMEC apoptosis,decreased MDA content,increased SOD and CAT activities,decreased the relative expression of Bax protein and increased the re

关 键 词：肺癌转移相关转录本1 miR-532-3p 脑微血管内皮细胞 凋亡 氧化损伤 

分 类 号：R363[医药卫生—病理学]