[1]袁会军,芦　军,崔志强,等.微RNA-543对过氧化氢诱导的人静脉内皮细胞氧化应激的影响[J].新乡医学院学报,2021,38(3):233-239.[doi:10.7683/xxyxyxb.2021.03.006]
　YUAN Huijun,LU Jun,CUI Zhiqiang,et al.Effect of microRNA-543 on hydrogen peroxide-induced oxidative stress in human venous endothelial cells[J].Journal of Xinxiang Medical University,2021,38(3):233-239.[doi:10.7683/xxyxyxb.2021.03.006]
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微RNA-543对过氧化氢诱导的人静脉内皮细胞氧化应激的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
38
期数:
2021年3
页码:
233-239
栏目:
基础研究
出版日期:
2021-03-05

文章信息/Info

Title:
Effect of microRNA-543 on hydrogen peroxide-induced oxidative stress in human venous endothelial cells
作者:
袁会军; 芦　军; 崔志强; 韩　鹏
（西安交通大学附属红会医院介入周围血管科，陕西　西安　710054）
Author(s):
YUAN Huijun; LU Jun; CUI Zhiqiang; HAN Peng
（Radioactive Interventional Division，Honghui Hospital Affiliated to Xi′an Jiaotong University,Xi′an 710054，Shaanxi Province，China）
关键词:
微RNA-543; 内皮细胞损伤; 氧化应激; 炎症因子; 血栓形成相关因子
Keywords:
microRNA-543endothelial cell injuryoxidative stressinflammatory factorsthrombosis-related factors
分类号:
R543.3
DOI:
10.7683/xxyxyxb.2021.03.006
文献标志码:
A
摘要:
目的　探讨微RNA-543(miR-543）对过氧化氢（H2O2）诱导的人静脉内皮细胞（HUVECs）氧化应激的影响。方法　将对数生长期的HUVECs 随机分为对照组及100、200、300、400 μmol·L-1H2O2组。对照组细胞于达尔伯克改良伊格尔培养基中常规培养；100、200、300、400 μmol·L-1H2O2组细胞分别加入终浓度100、200、300、400 μmol·L-1 H2O2诱导培养各组细胞培养3 h时采用四甲基偶氮唑盐(MTT)法检测细胞活性。另设inhibitor+H2O2组和inhibitor-NC+H2O2组。inhibitor+H2O2组HUVECs转染miR-543 inhibitor后，再用300 μmol·L-1H2O2诱导培养3 h；inhibitor-NC+H2O2组HUVECs转染inhibitor-NC后，再用300 μmol·L-1H2O2诱导培养3 h。取对照组、300 μmol·L-1H2O2组、inhibitor+H2O2组和inhibitor-NC+H2O2组HUVECs，采用MTT法检测细胞活性，实时荧光定量聚合酶链反应法检测细胞中miR-543水平，酶标仪法检测细胞中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性及丙二醛（MDA）和一氧化氮（NO）水平，Western blot法检测细胞上清液中白细胞介素-1β（IL-1β）、细胞间黏附分子（ICAM1）、血管细胞黏附分子（VCAM1）、血栓调节蛋白（TM）、组织因子途径抑制因子（TFPI）和内皮型一氧化氮合酶（eNOS）的相对表达量；双荧光素酶报告基因法检测HUVECs中荧光素酶活性。结果　与对照组比较，300 μmol·L-1H2O2组、inhibitor+H2O2组和inhibitor-NC+H2O2组HUVECs细胞活性显著降低，miR-543相对表达量显著增高，SOD和GSH-Px活性、NO水平以及TM、TFPI、eNOS蛋白相对表达量显著降低，MDA水平及IL-1β、ICAM1和VCAM1蛋白相对表达量显著升高（P<0.05）。与300 μmol·l-1H2O2组和inhibitor-NC+H2O2组比较，inhibitor+H2O2组HUVECs细胞活性显著升高，miR-543相对表达量显著降低，SOD和GSH-Px活性、NO水平以及TM、TFPI、eNOS蛋白相对表达量显著升高，MDA水平及IL-1β、ICAM1和VCAM1蛋白相对表达量显著降低（P<0.05）。300 μmol·l-1H2O2组与inhibitor-NC+H2O2组HUVECs细胞活性和miR-543相对表达量、SOD和GSH-Px活性、NO、MDA水平及TM、TFPI、eNOS、IL-1β、ICAM1和VCAM1蛋白相对表达量比较差异均无统计学意义（P>0.05）。miR-543 mimic+pGL3-TFPI 3′-UTR组荧光素酶活性显著低于miR-543 mimic+pGL3-TFPI 3′-UTR mut组（P<0.05）；mir-543 mimic+pgl3-enos="" 3′-utr组荧光素酶活性显著低于mir-543="" 3′-utr="" mut组（P<0.05）。mir-543 mimic组mir-543表达高于对照组和mimic-nc组（P<0.05）；mir-543 mimic组tfpi和enos相对表达量显著低于对照组和mimic-nc组（P<0.05）；对照组与mimic-nc组mir-543、tfpi和enos相对表达量比较差异均无统计学意义（>P>0.05）。结论　抑制miR-543表达能够抵抗H2O2诱导的HUVECs损伤，提高细胞活性、抑制氧化应激和炎症因子水平，提高抗血栓能力。undefinedundefinedundefinedundefinedundefinedundefined/i="">/sup="">/html>
Abstract:
Objective　To investigate the effects of microRNA-543 (miR-543) on oxidative stress，inflammatory response and thrombosis-related factors in human vascular endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2).Methods　The HUVECs in logarithmic growth phase were randomly divided into control group and 100，200，300,400 μmol·L-1 H2O2 groups.The cells in control group were cultured in Dulbecco′s modified Eagle′s medium.The cells in the 100，200，300,400 μmol·L-1 H2O2 groups were added with the final concentration of 100，200，300 and 400 μmol·L-1 H2O2 for induction culture，respectively；the HUVECs in each group were cultured for 3 h，and cell viability was detected by methyl thiazolyltetrazolium (MTT) assay.Inhibitor+H2O2 group and inhibitor-NC+H2O2 group were also set up.After transfected with miR-543 inhibitor，HUVECs in inhibitor+H2O2 group were induced by 300 μmol·L-1 H2O2 for 3 h.HUVECs in inhibitor-NC+H2O2 group were transfected with inhibitor-NC and then induced by 300 μmol·L-1 H2O2 for 3 h.HUVECs in the control group，300 μmol·L-1 H2O2 group，inhibitor+H2O2 group and inhibitor-NC+H2O2 group were taken，and the cell viability was detected by MTT assay，miR-543 level was detected by real-time quantitative polymerase chain reaction，and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)，the levels of malondialdehyde (MDA) and nitric oxide (NO) were detected by microplate reader method，the relative expression levels of interleukin (IL)-1β，intercellular adhesion molecule (ICAM1)，vascular cell adhesion molecule (VCAM1)，thromboregulatory protein (TM)，tissue factor pathway inhibitor (TFPI) and endothelial nitric oxide synthase (eNOS) were detected by Western blot.The luciferase activitng of HUVECS wasdetected by luciferase reporter assay.Results　Compared with the control group，the cell viability of HUVECs in 300 μmol·L-1 H2O2 group，inhibitor+H2O2 group and inhibitor-NC+H2O2 group was significantly decreased，the relative expression level of miR-543 was markedly increased，the levels of SOD，GSH-Px，NO and the relative expression levels of TM，TFPI，eNOS protein were significantly decreased，the level of MDA and the relative expression levels of IL-1β，ICAM1 and VCAM1 protein were significantly increased (P<0.05).Compared with 300 μmol·L-1 H2O2 group and inhibitor-NC+H2O2 group，the cell viability of HUVECs in inhibitor+H2O2 group was significantly increased，the relative expression level of miR-543 was markedly decreased，the activity of SOD and GSH-Px，the levels of NO and the relative expression levels of TM，TFPI，eNOS protein were significantly increased，the level of MDA and the relative expression levels of IL-1β，ICAM1 and VCAM1 protein were significantly decreased (P<0.05) .There were no significant differences in the cell viability,the relative expression levels of miR-543，the activity of SOD and GSH-Px，the levels of NO and MDA，the relative expression levels of TM，TFPI，eNOS，IL-1β，ICAM1 and VCAM1 protein in HUVECs between the 300 μmol·L-1 H2O2 group and inhibitor-NC+H2O2 group (P>0.05).the luciferase activity in the miR-543 mimic+pGL3-TFPI 3′-UTR group was significantly lower than that in the miR-543 mimic+pGL3-TFPI 3′-UTR mut group (P<0.05);the luciferase activity in the miR-543 mimic+pGL3-eNOS 3′-UTR group was significantly lower than that in the miR-543 mimic+pGL3-eNOS 3′-UTR mut group (P<0.05).The expression level of miR-543 in the miR-543 mimic group was significantly higher than that in the control group and mimic-NC group (P<0.05);the relative expression levels of TFPI and eNOS in the miR-543 mimic group were significantly lower than those in the control group and mimic-NC group (P<0.05);there was no significant difference in the expression level of miR-543，TFPI and eNOS between the control group and mimic-NC group(P>0.05).Conclusion　Inhibition of miR-543 expression could be resistant to H2O2-induced HUVECs injury，increase cell viability，inhibit oxidative stress and inflammatory factor levels，and elevate antithrombotic ability.

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更新日期/Last Update: 2021-03-05