miR-181a过表达加重H_2O_2诱导的HUVECs损伤
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摘要
目的研究miR-181a在过氧化氢(H_2O_2)损伤的人脐静脉内皮细胞(HUVECs)中的表达,并探讨miR-181a对H_2O_2诱导的HUVECs活性和凋亡的影响及其机制。方法 MTT法和流式细胞计量术测定HUVECs活性和凋亡;RT-qCR和Western blot测定miR-181a和X连锁凋亡抑制蛋白(XIAP)mRNA及蛋白的表达;Targetscan软件预测miR-181a和XIAP的靶向关系,利用双荧光素酶报告分析和Western blot加以验证。结果 1)H_2O_2呈剂量依赖性抑制HUVECs活性并促进凋亡(P<0.05);2)miR-181a在H_2O_2处理的HUVECs中显著升高(P<0.05);3)敲低miR-181a可明显促进H_2O_2诱导的HUVECs活性并抑制细胞凋亡(P<0.05);4)miR-181a能够与XIAP靶向结合;5)miR-181a过表达可抑制H_2O_2诱导的HUVECs活性并促进细胞凋亡,外源过表达XIAP可减轻miR-181a调控的HUVECs活性和凋亡。结论 miR-181a通过靶向XIAP抑制H_2O_2诱导的HUVECs活性,并促进细胞凋亡,加重HUVECs损伤。
Objective To investigate the expression of miR-181 a in hydrogen peroxide(H_2O_2)-injured human umbilical vein endothelial cells(HUVECs) by H_2O_2, and the effect and mechanism of miR-181 a on cell viability and apoptosis. Methods The viability and apoptosis of HUVECs were measured by MTT and flow cytometry assays. miR-181 a and X-linked inhibitor of apoptosis(XIAP) expressions were determined by RT-qCR and Western blot analyses. The binding sites of miR-181 a in XIAP 3′UTR were predicted using Targetscan software and verified by Dual-Luciferase Reporter and Western blot assays. Results 1)H_2O_2 inhibited cell activity while promoted apoptosis of HUVECs in a dose-dependent manner(P<0.05). 2)miR-181 a was significantly elevated in H_2O_2-treated HUVECs(P<0.05). 3)Knockdown of miR-181 a promoted H_2O_2-induced cell activity and inhibited apoptosis(P<0.05). 4)XIAP was identified to be a target for miR-181 a. 5)Overexpression of miR-181 a inhibited H_2O_2-induced cell activity and promoted apoptosis, which was reversed by XIAP restoration. Conclusions miR-181 a stimulates H_2O_2-induced cell activity while inhibites cell apoptosis by targeting at XIAP and then leading to the aggravation of HUVECs injury.
Objective To investigate the expression of miR-181 a in hydrogen peroxide(H_2O_2)-injured human umbilical vein endothelial cells(HUVECs) by H_2O_2, and the effect and mechanism of miR-181 a on cell viability and apoptosis. Methods The viability and apoptosis of HUVECs were measured by MTT and flow cytometry assays. miR-181 a and X-linked inhibitor of apoptosis(XIAP) expressions were determined by RT-qCR and Western blot analyses. The binding sites of miR-181 a in XIAP 3′UTR were predicted using Targetscan software and verified by Dual-Luciferase Reporter and Western blot assays. Results 1)H_2O_2 inhibited cell activity while promoted apoptosis of HUVECs in a dose-dependent manner(P<0.05). 2)miR-181 a was significantly elevated in H_2O_2-treated HUVECs(P<0.05). 3)Knockdown of miR-181 a promoted H_2O_2-induced cell activity and inhibited apoptosis(P<0.05). 4)XIAP was identified to be a target for miR-181 a. 5)Overexpression of miR-181 a inhibited H_2O_2-induced cell activity and promoted apoptosis, which was reversed by XIAP restoration. Conclusions miR-181 a stimulates H_2O_2-induced cell activity while inhibites cell apoptosis by targeting at XIAP and then leading to the aggravation of HUVECs injury.
引文
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[3] Zhang P,Xu,Li XC.Cardiovascular diseases:oxidative damage and antioxidant protection [J].Eur Rev Med Pharmacol Sci,2014,18:3091-3096.
[4] Chen CZ,Li L,Lodish HF,et al.MicroRNAs modulate hematopoietic lineage differentiation [J].Science,2004,303:83-86.
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[7] 秦冰,肖波,姜婷,等.MiR-590-5p对OX-LDL诱导血管内皮细胞凋亡以及LOX-1表达的影响[J].中南大学学报(医学版),2012,37:675-681.
[8] Hao X,Kang Y,Li J,et al.Protective effects of hyperoside against H2O2-induced apoptosis in human umbilical vein endothelial cells [J].Mol Med Rep,2016,14:399-405.
[9] Lee R,Margaritis M,Channon KM,et al.Evaluating oxidative stress in human cardiovascular disease:methodological aspects and considerations [J].Curr Med Chem,2012,19:2504-2520.
[10] Magenta A,Cencioni C,Fasanaro P,et al.miR-200c is upregulated by oxidative stress and induces endothelial cell apoptosis and senescence via ZEB1 inhibition [J].Cell Death Differ,2011,18:1628-1639.
[11] 李添祎.microRNA-210在H2O2诱导的内皮细胞和心肌细胞凋亡中的作用及机制研究 [D].吉林大学,2017.
[12] Yan X,Tan Y,Yuan Y,et al.Upregulation of miR-181a-5p represses VEGF pathway in high glucose treated human retinal endothelial cells [J].Int J Clin Exp Med,2016,9:2493-2499.
[13] Liu G,Li Y,Gao XG.microRNA-181a is upregulated in human atherosclerosis plaques and involves in the oxida-tive stress-induced endothelial cell dysfunction through direct targeting Bcl-2 [J].Eur Rev Med Pharmacol Sci,2016,20:3092-3100.
[14] Shin S,Moon KC,Park KU,et al.MicroRNA-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of X-linked inhibitor of apoptotic protein in endothelial cells [J].Biochimie,2012,94:1431-1436.
[15] Long SY,Liu Y,Yang Y,et al.TSG up-regulate XIAP and inhibit apoptosis of human umbilical vein endothelial cells [J].Progress in Modern Biomedicine,2014,19:3606-3610.
[16] Prager GW,Mihaly J,Brunner PM,et al.Urokinase mediates endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein [J].Blood,2009,113:1383-1390.