| 282 | 1 | 23 |
| 下载次数 | 被引频次 | 阅读次数 |
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。
Abstract：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".
[1] LIBBY P.The changing landscape of atherosclerosis[J].Nature,2021,592(7855):524-533.
[2] ROHLENOVA K,VEYS K,MIRANDA-sANTOS I,et al.Endothelial cell metabolism in health and disease[J].Trends in Cell Biology,2018,28(3):224-236.
[3] BACH D,LEE S,SOOD A.Circular RNAs in cancer[J].Molecular Therapy-Nucleic Acids,2019,16:118-129.
[4] SEKAR S,LIANG W.Circular RNA expression and function in the brain[J].Non-coding RNA Research,2019,4(1):23-29.
[5] KAWAGUCHI S,MOUKETTE B,HAYASAKA T,et al.Noncoding RNAs as key regulators for cardiac development and cardiovascular diseases[J].Journal of Cardiovascular Development and Disease,2023,10(4):166.
[6] DANG R,LIU F,Li Y.Circular RNA hsa_circ_0010729 regulates vascular endothelial cell proliferation and apoptosis by targeting the miR-186/HIF-1alpha axis[J].Biochemical and Biophysical Research Communications,2017,490(2):104-110.
[7] MUMTAZ P,TABAN Q,DAR M,et al.Deep insights in circular RNAs:from biogenesis to therapeutics[J].Biological Procedures Online,2020,22:10.
[8] MA S,KONG S,WANG F,et al.CircRNAs:biogenesis,functions,and role in drug-resistant tumours[J].Molecular Cancer,2020,19(1):119.
[9] ZHONG Y,DUI Y,YANG X,et al.Circular RNAs function as ceRNAs to regulate and control human cancer progression[J].Molecular Cancer,2018,17(1):79.
[10]XUE D,WANG H,CHEN Y,et al.Circ-AKT3 inhibits clear cell renal cell carcinoma metastasis via altering miR-296-3p/E-cadherin signals[J].Molecular Cancer,2019,18(1):151.
[11]WANG L,ZHENG Z,FENG X,et al.circRNA/lncRNA-miRNA-mRNA network in oxidized,low-density,lipoprotein-induced foam cells[J].DNA and Cell Biology,2019,38(12):1499-1511.
[12]VO J,CIESIIK M,ZHANG Y,et al.The landscape of circular RNA in cancer[J].Cell,2019,176(4):869-881.
[13]KRISTENSEN L,ANDERSEN M,STAGSTED L,et al.The biogenesis,biology and characterization of circular RNAs[J].Nature Reviews Genetics,2019,20(11):675-691.
[14]LI X,YANG L,CHEN L.The biogenesis,functions,and challenges of circular RNAs[J].Molecular Cell,2018,71(3):428-442.
[15]LIANG W,WONG C,LIANG P,et al.Translation of the circular RNA circβ-catenin promotes liver cancer cell growth through activation of the Wnt pathway[J].Genome Biology,2019,20(1):84.
[16]CHEKULAEVA M,RAJEWSKY N.Roles of long noncoding RNAs and circular RNAs in translation[J].Cold Spring Harbor Perspectives in Biology,2019,11(6):a032680.
[17]LEE S,CHEN T,BARBER C L,et al.Autocrine VEGF signaling is required for vascular homeostasis[J].Cell,2007,130(4):691-703.
[18]YAN D,ZHANG D,LU L,et al.Vascular endothelial growth factor-modified macrophages accelerate reendothelialization and attenuate neointima formation after arterial injury in atherosclerosis-prone mice[J].Journal of Cellular Biochemistry,2019,120(6):10652-10661.
[19]JIANG Z,JIANG Y.Circular RNA circPDS5B impairs angiogenesis following ischemic stroke through its interaction with hnRNPL to inactivate VEGF-A[J].Neurobiology of Disease,2023,181:106080.
[20]ZHANG Y,ZHAO Y,YU F,et al.CircRNA_06354 might promote early-onset preeclampsia in humans via hsa-miR-92a-3p/vascular endothelial growth factor-A[J].Journal of Hypertension,2023,41(3):494-507.
基本信息:
DOI：10.19367/j.cnki.2096-8388.2024.01.012
中图分类号:R543.5
引用信息:
[1]陈万,王小静,刘晗,等.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[J].贵州医科大学学报,2024,49(01):90-95.DOI:10.19367/j.cnki.2096-8388.2024.01.012.
基金信息:
重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0919)
2024-01-28
2024-01-28