沉默LncRNA MEG3调控miR-424-5p/FoxO1对氧化型低密度脂蛋白诱导的动脉粥样硬化的保护机制  

Silencing LncRNA MEG3 regulates the protective mechanism of miR-424-5p/FoxO1 against oxidized low-density lipoprotein-induced atherosclerosis

作　　者：任丽 吴锡骅 刘婷 梅益彰 Ren Li;Wu Xihua;Liu Ting;Mei Yizhang(Department of Neurology,Foxing Chancheng Hospital,Foshan 528031,China)

机构地区：[1]广东佛山复星禅诚医院神经内科,佛山528031

出　　处：《中华细胞与干细胞杂志（电子版）》2022年第6期335-345,共11页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：广东省佛山市科技攻关项目(1920001000474)。

摘　　要：目的探究LncRNA MEG3在动脉粥样硬化(AS)发展中的作用及潜在机制。方法培养人脐静脉内皮细胞(HUVECs),采用氧化型低密度脂蛋白(ox-LDL)诱导建立内皮细胞损伤模型。ox-LDL干预HUVECs为ox-LDL组;分别用sh-NC、sh-MEG3、sh-MEG3和in-miR-424-5p、sh-MEG3和oe-FoxO1转染后ox-LDL干预HUVECs设为ox-LDL+sh-NC组、ox-LDL+sh-MEG3组、ox-LDL+sh-MEG3+in-miR-424-5p组、ox-LDL+sh-MEG3+oe-FoxO1组;ox-LDL相同的溶剂处理HUVECs为对照。采用qRT-PCR法检测LncRNA MEG3、miR-424-5p和FoxO1 mRNA表达水平;采用CCK-8和EdU法检测细胞增殖;采用流式细胞术检测细胞凋亡;小管形成实验检测血管生成;Western blot法检测FoxO1蛋白、血管生成相关蛋白NOS3、VEGFA和CXCL12的水平;双荧光素酶实验检测LncRNA MEG3和miR-424-5p靶标关系;建立AS小鼠模型,油红O染色检测小鼠AS病变面积;伊文思蓝染色评估血管再内皮化程度。两组间比较采用t检验,多组间比较采用单因素方差分析,进一步两两比较采用SNK-q检验。结果与对照比较,ox-LDL组LncRNA MEG3表达(2.35±0.24比1.03±0.04)、FoxO1 mRNA和蛋白水平及细胞凋亡率上升(P均<0.05);miR-424-5p表达(0.37±0.05比1.03±0.08)、细胞活力、EdU阳性细胞数、小管管型分枝数、NOS3、VEGFA和CXCL12蛋白水平均下降(P均<0.05)。与ox-LDL+sh-NC组比较,ox-LDL+sh-MEG3组LncRNA MEG3表达、FoxO1 mRNA和蛋白水平及细胞凋亡率下降;miR-424-5p表达、细胞活力、EdU阳性细胞数、小管管型分枝数及NOS3、VEGFA和CXCL12蛋白水平均上升(P均<0.05)。抑制miR-424-5p或过表达FoxO1可部分逆转LncRNA MEG3沉默对ox-LDL刺激HUVECs的影响(P<0.05)。LncRNA MEG3通过miR-424-5p靶向调控FoxO1表达(P<0.05)。在体内,AS小鼠颈主动脉中LncRNA MEG3、FoxO1 mRNA和蛋白表达升高,而miR-424-5p、NOS3、VEGFA和CXCL12蛋白表达下调;同时AS病变面积增加,血管再内皮化受到抑制(P<0.05)。沉默LncRNA MEG3后可改善上述AS小鼠的典型特征。结论沉默LncRNAObjective To explore the role and potential mechanism of LncRNA MEG3 in developing atherosclerosis(AS).Methods Human umbilical vein endothelial cells(HUVECs)were cultured,and oxidized low-density lipoprotein(ox-LDL)was used to induce the endothelial cell injury model.The experiment was divided into a control group,ox-LDL group,ox-LDL+sh-NC group,ox-LDL+sh-MEG3 group,ox-LDL+sh-MEG3+in-miR-424-5p group,ox-LDL+sh-MEG3+oe-FoxO1 group.The expression levels of LncRNA MEG3,miR-424-5p and FoxO1 mRNA were detected by qRT-PCR;CCK-8 and EdU methods were used to detect cell proliferation;apoptosis was detected by flow cytometry;angiogenesis was detected by tubule formation assay;the levels of FoxO1 protein,angiogenesis related proteins NOS3,VEGFA and CXCL12 were determined by Western blot;dual luciferase assay detection of LncRNA MEG3 and miR-424-5p target relationship.The AS mouse model was established,and oil red O staining was used to detect the area of AS lesions in mice;Evans blue staining was used to evaluate the degree of vascular re-endothelialization.The t-test was used for comparison between two groups;the one-way analysis of variance was used for multiple groups,and the SNK-q test was used for further pairwise comparison.Results Compared with the control group,LncRNA MEG3 expression(2.35±0.24 vs 1.03±0.04),FoxO1 mRNA and protein levels,and apoptosis rate were increased after ox-LDL group(P<0.05);miR-424-5p expression(0.37±0.05 vs 1.03±0.08),cell viability,number of EdU positive cells,the number of tubule branching,NOS3,VEGFA and CXCL12 protein levels were all decreased(P<0.05).Compared with the ox-LDL+sh-NC group,LncRNA MEG3 expression,FoxO1 mRNA and protein levels and cell apoptosis rate in ox-LDL+sh-MEG3 group decreased(P<0.05);miR-424-5p expression(P<0.05),cell viability,number of EdU positive cells,number of tubule branching and protein levels of NOS3,VEGFA and CXCL12 were all increased(P<0.05).Inhibition of miR-424-5p or overexpression of FoxO1 partially reversed the effect of LncRNA MEG3 silencing on ox-

关 键 词：LncRNA MEG3 动脉粥样硬化 增殖 凋亡 血管生成 

分 类 号：R543.5[医药卫生—心血管疾病]