氧化低密度脂蛋白对人脐静脉内皮细胞增殖及circRNA-CER表达的影响  

Effects of oxidized lipoprotein on the proliferation of human umbilical vein endothelial cells and the expression of circRNA-CER

作　　者：陈万 王小静 刘晗 赵郑波 CHEN Wan;WANG Xiaojing;LIU Han;ZHAO Zhengbo(Department of Cardiology,People's Hospital of Jiulongpo District of Chongqing,Chongqing,400050,China;Department of Neurology,People's Hospital of Jiulongpo District of Chongqing,Chongqing,400050,China;Department of Cardiology,traditional Chinese Medicine Hospital of Yubei District of Chongqing,Chongqing 401121,China)

机构地区：[1]重庆市九龙坡区人民医院心内科,重庆400050 [2]重庆市九龙坡区人民医院神经内科,重庆400050 [3]重庆市渝北区中医院心内科,重庆401121

出　　处：《贵州医科大学学报》2024年第1期90-95,共6页Journal of Guizhou Medical University

基　　金：重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0919)。

摘　　要：目的探讨氧化低密度脂蛋白(ox-LDL)刺激人脐静脉内皮细胞(HUVEC)后环状RNA circRNA-CER(circRNA-100876)的表达及其对HUVEC增殖的影响。方法分别以不同浓度ox-LDL(40、60、80、100及200 mg/L)刺激HUVEC 24 h,以及以100 mg/L ox-LDL刺激HUVEC 6、12、24及48 h,利用实时荧光定量PCR验证circRNA-CER在细胞中的表达水平;构建circRNA-CER特异性过表达质粒并进行细胞转染,采用CCK8实验研究circRNA-CER在血管内皮细胞增殖中的作用;荧光定量PCR检测circRNA-CER对miR-136表达的影响;Western blot检测过表达circRNA-CER后血管内皮生长因子(VEGF)的蛋白表达水平,荧光素酶报告基因验证circRNA-CER与miR-136、miR-136与VEGF-3′UTR的相互作用。结果随ox-LDL处理浓度增加,circRNA-CER在HUVEC中的表达量呈下调趋势,并在100 mg/L时差异有统计学意义(P<0.05);在用该浓度ox-LDL以时间梯度刺激HUVEC后,circRNA-CER表达随刺激时间的延长而下调,在24 h处差异有统计学意义(P<0.05);上调circRNA-CER表达可抑制ox-LDL(100 mg/L、24 h)对HUVEC增殖的抑制作用,上调miR-136的RNA及VEGF的蛋白表达水平,同时干预miR-136表达可阻止circRNA-CER对VEGF表达的调控;荧光素酶报告基因实验证实circRNA-CER与miR-136、miR-136可与VEGF 3′-UTR直接结合。结论circRNA-CER可阻止ox-LDL对HUVEC增殖的抑制效应,机制为circRNA-CER通过“海绵样”作用调控miR-136后,影响其对下游靶基因VEGF的转录后调控。Objective To investigate the effect of oxidized lipoprotein(ox-LDL)as it stimulated human umbilical vein endothelial cells(HUVEC)and the proliferation of HUVEC and the expression of circRNA-CER(circRNA-100876)in ox-LDL.Methods Real-time PCR was performed to examine circRNA-CER expression in HUVEC treated with ox-LDL(40,60,80,100,and 200 mg/L)for 24 h,and 100 mg/L ox-LDL for 6,12,24,and 48 h.The expression level of circRNA-CER in cells was verified by real-time quantitative PCR(RT-qPCR),and the specific overexpression plasmid of circRNA-CER in vitro was constructed and transfected into cells,the effect of circRNA-CER on HUVEC proliferation was analyzed by CCK8 assay,and the expression of miR-136 was detected by RT-qPCR;The protein expression of vascular endothelial growth factor(VEGF)was detected by Western blot,and the interaction of circRNA-CER with miR-136 and miR-136 with Vegf-3'UTR was confirmed by Luciferase reporter gene assay.Results circRNA-CER was downregulated upon ox-LDL treatment in a dose-dependent manner with significantly depressed expression at 100 mg/L(P<0.05).Likewise,the expression of circRNA-CER was inhibited in 100 mg/L ox-LDL treated HUVEC for indicated time,showing significant change 24 h after treatment(P<0.05).The overexpression of circRNA-CER suppressed the inhibition of ox-LDL(100 mg/L,24 h)on HUVEC proliferation and increased the RNA level of miR-136 and the protein level of VEGF.Concurrent interfering of miR-136 blocked the regulation of circRNA-CER on VEGF expression.Luciferase reporter gene assay verified the direct combination of circRNA-CER with miR-136 and miR-136 with VEGF-3'UTR.Conclusion circRNA-CER can inhibit the suppressing effect of ox-LDL on HUVEC proliferation.Mechanistically,circRNA-CER can affect post-transcriptional regulation of its target gene VEGF through regulating miR-136 via acting as a molecular"sponge".

关 键 词：环状RNA 微小RNA 动脉粥样硬化 人脐静脉内皮细胞 氧化低密度脂蛋白 

分 类 号：R285[医药卫生—中药学]