LncRNA NORAD通过miR-513b-5p/GREM1轴调节颅内动脉瘤血管平滑肌细胞增殖、迁移、侵袭和凋亡的作用机制  

LncRNA NORAD Regulates the Proliferation,Migration,Invasion,and Apoptosis of Intracranial Aneurysm Vascular Smooth Muscle Cells through the miR-513b-5p/GREM1 Axis

作　　者：黄锐[1] 陈海浚[1] 韦总当 秦国文[1] 钟书[1] 庞刚[1] HUANG Rui;CHEN Haijun;WEI Zongdang;QIN Guowen;ZHONG Shu;PANG Gang(The People′s Hospital of Guangxi Zhuang Autonomous Region,Nanning 530016,Guangxi,China)

机构地区：[1]广西壮族自治区人民医院,南宁530016

出　　处：《中西医结合心脑血管病杂志》2024年第15期2761-2769,共9页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：广西壮族自治区卫生健康委员会自筹经费科研课题(No.Z-A20220001)。

摘　　要：目的:探讨长链非编码RNA DNA损伤诱导的非编码RNA(LncRNA NORAD)通过miR-513b-5p/GREM1轴调节颅内动脉瘤血管平滑肌细胞(VSMC)增殖、迁移、侵袭和凋亡的机制。方法:采用实时荧光定量聚合酶链式反应(PCR)法检测人颅内动脉瘤组织和正常组织中LncRNA NORAD、miR-513b-5p及GREM1表达。体外分离培养人VSMC,随机分为对照组、LncRNA NORAD siRNA组、miR-513b-5p mimics组、共转染(LncRNA NORAD siRNA+miR-513b-5p inhibitor)组、共转染阴性对照(LncRNA NORAD siRNA阴性对照+miR-513b-5p inhibitor阴性对照)组,分组转染后,采用实时荧光定量PCR法检测各组细胞LncRNA NORAD、miR-513b-5p及GREM1 mRNA表达;采用细胞计数试剂盒(CCK-8)和免疫荧光染色检测各组细胞增殖情况;采用Hoechst 33342染色和免疫荧光染色检测各组细胞凋亡情况;采用细胞划痕实验和Transwell实验检测各组细胞迁移、侵袭情况;采用免疫印记实验检测各组细胞上皮间充质转化(EMT)标志蛋白神经钙黏素(N-cadherin)、E-钙黏素(E-cadherin)、波形蛋白(Vimentin)表达;采用双荧光素酶报告实验分析VSMC中LncRNA NORAD对miR-513b-5p、miR-513b-5p对GREM1的靶向调控。结果:与正常组织比较,颅内动脉瘤组织LncRNA NORAD、GREM1 mRNA表达明显升高(P<0.05),miR-513b-5p表达明显降低(P<0.05)。与对照组比较,LncRNA NORAD siRNA组、miR-513b-5p mimics组细胞GREM1 mRNA表达、增殖率、Ki67阳性率、迁移率、侵袭数及N-cadherin、Vimentin蛋白表达降低(P<0.05),miR-513b-5p表达、凋亡率及Bax/Bcl-2、E-cadherin蛋白表达升高(P<0.05);共转染阴性对照组各指标差异无统计学意义(P>0.05)。与LncRNA NORAD siRNA组比较,共转染组细胞GREM1 mRNA表达、增殖率、Ki67阳性率、迁移率、侵袭数及N-cadherin、Vimentin蛋白表达升高(P<0.05),miR-513b-5p表达、凋亡率及Bax/Bcl-2、E-cadherin蛋白表达降低(P<0.05)。结论:敲低LncRNA NORAD可通过上调miR-513b-5p表达而降低GREM1表达,从而抑制VSMObjective:To study the mechanism of long non-coding RNA DNA damage induced non-coding RNA(LncRNA NORAD)regulating the proliferation,migration,invasion,and apoptosis of vascular smooth muscle cells(VSMC)in intracranial aneurysm(IA)through the miR-513b-5p/GREM1 axis.Methods:Real-time fluorescence quantitative PCR was used to detect the expression of LncRNA NORAD,miR-513b-5p,and GREM1 in human intracranial aneurysm tissue and normal tissue.Human vascular smooth muscle cells(VSMCs)were isolated,cultured in vitro,and randomly separated into control group,LncRNA NORAD siRNA group,miR-513b-5p mimics group,co-transfection(LncRNA NORAD siRNA+miR-513b-5p inhibitor)group,and co-transfection negative control(LncRNA NORAD siRNA negative control+miR-513b-5p inhibitor negative control)group.After grouping and transfection,real-time fluorescence quantitative PCR was used to detect the expression of LncRNA NORAD,miR-513b-5p,and GREM1 mRNA of cells in each group.The proliferation of cells in each group was detected by CCK-8 method and immunofluorescence staining.Hoechst 33342 staining and immunofluorescence staining were used to detect cell apoptosis in each group.Cell migration and invasion in each group were detected by cell scratch assay and Transwell assay.The expression of EMT marker proteins N-cadherin,E-cadherin,and Vimentin of cells in each group was detected by western Blotting.Dual-luciferase reporter assay was used to analyze the targeting regulation of miR-513b-5p by LncRNA NORAD and the targeted regulation of GREM1 by miR-513b-5p in VSMC.Results:Compared with normal tissue,the expression of LncRNA NORAD and GREM1 mRNA in intracranial aneurysm tissue obviously increased(P<0.05),and the expression of miR-513b-5p obviously decreased(P<0.05).Compared with control group,the expression of GREM1 mRNA,proliferation rate,Ki67 positive rate,migration rate,invasion number,and the expression of N-cadherin and Vimentin proteins in cells decreased in LncRNA NORAD siRNA group and miR-513b-5p mimics group(P<0.05),the expression of mi

关 键 词：颅内动脉瘤 长链非编码RNA DNA损伤诱导的非编码RNA LncRNA NORAD miR-513b-5p GREM1 血管平滑肌细胞 实验研究 

分 类 号：R743[医药卫生—神经病学与精神病学]