环状RNA_0000231对血管平滑肌细胞增殖和迁移的影响  

Effects of circular RNA_0000231 on proliferation and migration of vascular smooth muscle cells

作　　者：苗仁英 王金亮 于朝洋 张一飞 付一群 吕程威 杨博[1] 宋燕[1] Miao Renying;Wang Jinliang;Yu Chaoyang;Zhang Yifei;Fu Yiqun;Lyu Chengwei;Yang Bo;Song Yan(Department of Vascular Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]河南郑州大学第一附属医院血管外科,郑州450000

出　　处：《中华实验外科杂志》2024年第11期2499-2504,共6页Chinese Journal of Experimental Surgery

基　　金：河南省自然科学基金项目(232300421179)。

摘　　要：目的:探讨环状RNA(circRNA)_0000231对血管平滑肌细胞(VSMCs)的增殖和迁移的影响及其调控机制。方法:选择24只北京协和医学院基础研究所的ApoE基因敲除(ApoE^(-/-))4周龄雄性C57BL/6J小鼠,将小鼠按数字表格法随机分为4组,每组6只,正常饮食组(normal组)给予普通饲料,作为空白对照、高脂饮食组(high fat组)给予高脂饲料,建立动脉粥样硬化(AS)小鼠模型、高脂饮食+空载体腺病毒组(Ad-sh-nc组)在高脂饮食基础上尾静脉注射搭载空载体腺病毒、高脂饮食+circRNA_0000231腺病毒敲低组(Ad-sh-circ)在高脂饮食基础上注射circRNA_0000231腺病毒敲低。12周后分别取各组小鼠的主动脉,检测circRNA_0000231的表达,采用血管油红O染色法检测AS程度。于37℃、5%CO_(2)条件下,在添加10%胎牛血清和1%青霉素/链霉素的高糖杜尔伯科改良伊格尔(DMEM)培养基中培养小鼠主动脉平滑肌细胞(MASMCs)以评估circRNA_0000231对MASMCs表型的影响。采用RNA下拉实验和双荧光素酶报告基因实验验证circRNA_0000231的下游调控靶基因。通过噻唑蓝(MTT)和细胞迁移实验(Transwell)检测circRNA_0000231对MASMCs增殖和迁移的影响。组间比较采用非配对t检验或非参数检验,对于3组或多组的多重比较采用单因素方差分析。结果:circRNA_0000231在AS模型的斑块组织中的表达高于normal组(1.03±0.06比4.98±1.37,t=7.042,P<0.01);血管油红O染色可见Ad-sh-circ组小鼠AS病变面积低于Ad-sh-nc组[(0.59±0.11)cm^(2)比(0.34±0.07)cm^(2),t=4.694,P<0.01],Ad-sh-circ组小鼠的斑块占比低于Ad-sh-nc组[(46.02±10.14)%比(21.05±8.81)%,t=4.552,P<0.01]。敲低circRNA_0000231可增加微小RNA(miR)-630的表达(0.99±0.03比3.77±0.19,t=24.824,P<0.01),而过表达circRNA_0000231可抑制miR-630的表达(0.99±0.12比0.41±0.05,t=7.695,P<0.01)。MTT结果表明circRNA_0000231的敲低抑制MASMCs的增殖(1.55±0.09比0.89±0.11,t=7.787,P<0.01)。Transwell和划痕实验表明,敲低circRNA_0000Objective To investigate the effects of circular RNA(circRNA)_0000231 on the proliferation and migration of vascular smooth muscle cells(VSMCs)and its regulatory mechanism.Methods Four-week-old male C57BL/6J mice with ApoE knockout(ApoE^(-/-))were used(24 mice were purchased from the Basic Research Institute of Peking Union Medical College,Beijing).The mice were randomly divided into four groups according to the digital table method:the normal diet group(normal group)was given ordinary feed as a blank control;The high-fat diet group(high fat group)was given a high-fat diet to establish an atherosclerosis(AS)mouse model;the high-fat diet+empty vector adenovirus group(Ad-sh-nc group)was injected with adenovirus carrying an empty vector through the tail vein on the basis of a high-fat diet;the high-fat diet+circRNA_0000231 adenovirus knockdown group(Ad-sh-circ)was injected with circRNA_0000231 adenovirus knockdown on the basis of a high-fat diet.After 12 weeks,the aortas of the mice were taken to detect the expression of circRNA_0000231,and the degree of atherosclerosis was detected by vascular oil red O staining.Mouse aortic smooth muscle cells(MASMCs)were cultured to evaluate the effect of circRNA_0000231 on their phenotypes.The medium(containing 10%FBS)was added,shaken,and placed in a 37℃incubator.RNA pull-down assay and dual luciferase reporter gene assay were used to verify the downstream regulatory target genes of circRNA_0000231.In addition,the effects of circRNA_0000231 on the proliferation and migration of MASMCs were detected by methyl thiazolyl tetrazolium(MTT)colorimetry and Transwell assay.Unpaired t-test or nonparametric test was used for comparison between groups,and one-way analysis of variance was used for multiple comparisons of three or more groups.ResultsThe expression of circRNA_0000231 in the plaque tissue of AS model mice was higher than that of normal mice(1.03±0.06 vs.4.98±1.37,t=7.042,P<0.01);oil red O staining of blood vessels showed that the area of atherosclerotic lesions in the expe

关 键 词：环状RNA_0000231 血管平滑肌细胞 动脉粥样硬化 微小RNA-630 

分 类 号：R54[医药卫生—心血管疾病]