miR-20a-5p通过靶向MRTFA缓解ox-LDL诱导的内皮细胞损伤
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摘要
目的探讨miR-20a-5p是否可通过靶向心肌素相关转录因子A(MRTFA)缓解氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)损伤。方法 RT-qPCR检测miR-20a-5p在不同剂量不同时间oxLDL诱导的HUVEC中的表达; MTT法、流式细胞术和Western blot检测过表达miR-20a-5p和MRTFA对ox-LDL诱导HUVEC增殖和凋亡的影响;乳酸脱氢酶(LDH)测试盒测定过表达miR-20a-5p对ox-LDL诱导HUVEC中LDH释放的影响; ELISA法检测过表达miR-20a-5p和MRTFA对ox-LDL处理的HUVEC中氧化应激指标超氧化物歧化酶(SOD)、一氧化氮(NO)、内皮型一氧化氮合酶(e NOS)和丙二醛(MDA)的影响;荧光素酶报告和Western blot实验验证miR-20a-5p与MRTFA的靶向关系。结果 miR-20a-5p的表达随着ox-LDL诱导时间和剂量的增加而逐渐下降;过表达miR-20a-5p可部分逆转ox-LDL诱导对HUVEC增殖和凋亡的影响;上调miR-20a-5p可部分修复ox-LDL诱导对HUVEC氧化应激损伤; MRTFA是miR-20a-5p的靶基因;在ox-LDL诱导HUVEC中,MRTFA过表达可逆转miR-20a-5p对ox-LDL诱导HUVEC细胞增殖和凋亡、氧化应激损伤指标的影响。结论 miR-20a-5p靶向MRTFA修复ox-LDL诱导的HUVEC损伤。
Aim To investigate whether microRNA-20 a-5 p( miR-20 a-5 p) alleviates oxidized low density lipoprotein( ox-LDL)-induced injury of human umbilical vein endothelial cells( HUVEC) by targeting myocardin-related transcription factor A( MRTFA). Methods RT-qPCR was used to detect the expression of miR-20 a-5 p in HUVEC induced by ox-LDL of various doses at different time. MTT,flow cytometry and Western blot assays were performed to evaluate the effect of overexpression of miR-20 a-5 p and MRTFA on the ox-LDL-induced proliferation and apoptosis of HUVEC.Lactate dehydrogenase( LDH) kit was conducted to determine the effect of overexpression of miR-20 a-5 p on ox-LDL-induced LDH release in HUVEC. ELISA assay was employed to examine the effects of upregulation of miR-20 a-5 p and MRFA on ox-LDL-mediated changes of oxidative stress indexes superoxide dismutase( SOD),nitric oxide( NO),endothelial nitric oxide synthasee( e NOS) and malondialdehyde( MDA) in HUVEC. Luciferase reporter Western blot assays was introduced to validate the relationship between miR-20 a-5 p and MRTFA. Results The expression of miR-20 a-5 p was significantly decreased in a time or dose dependent manner. Overexpression of miR-20 a-5 p attenuated the effect of oxLDL on proliferation,apoptosis,and LDH release in HUVEC. Upregulation of miR-20 a-5 p repaired ox-LDL-induced oxidative stress injury in HUVEC. MRTFA was a direct target gene of miR-20 a-5 p. MRTFA overexpression undermined the effects of miR-20 a-5 p on proliferation,apoptosis,and oxidative stress injury index in ox-LDL-treated HUVEC.Conclusion miR-20 a-5 p can repair ox-LDL-induced HUVEC damage via targeting MRTFA.
Aim To investigate whether microRNA-20 a-5 p( miR-20 a-5 p) alleviates oxidized low density lipoprotein( ox-LDL)-induced injury of human umbilical vein endothelial cells( HUVEC) by targeting myocardin-related transcription factor A( MRTFA). Methods RT-qPCR was used to detect the expression of miR-20 a-5 p in HUVEC induced by ox-LDL of various doses at different time. MTT,flow cytometry and Western blot assays were performed to evaluate the effect of overexpression of miR-20 a-5 p and MRTFA on the ox-LDL-induced proliferation and apoptosis of HUVEC.Lactate dehydrogenase( LDH) kit was conducted to determine the effect of overexpression of miR-20 a-5 p on ox-LDL-induced LDH release in HUVEC. ELISA assay was employed to examine the effects of upregulation of miR-20 a-5 p and MRFA on ox-LDL-mediated changes of oxidative stress indexes superoxide dismutase( SOD),nitric oxide( NO),endothelial nitric oxide synthasee( e NOS) and malondialdehyde( MDA) in HUVEC. Luciferase reporter Western blot assays was introduced to validate the relationship between miR-20 a-5 p and MRTFA. Results The expression of miR-20 a-5 p was significantly decreased in a time or dose dependent manner. Overexpression of miR-20 a-5 p attenuated the effect of oxLDL on proliferation,apoptosis,and LDH release in HUVEC. Upregulation of miR-20 a-5 p repaired ox-LDL-induced oxidative stress injury in HUVEC. MRTFA was a direct target gene of miR-20 a-5 p. MRTFA overexpression undermined the effects of miR-20 a-5 p on proliferation,apoptosis,and oxidative stress injury index in ox-LDL-treated HUVEC.Conclusion miR-20 a-5 p can repair ox-LDL-induced HUVEC damage via targeting MRTFA.
引文
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[18]Bian H,Lin JZ,Li C,et al. Myocardin-related transcription factor A(MRTFA)regulates the fate of bone marrow mesenchymal stem cells and its absence in mice leads to osteopenia[J]. Mol Metab,2016,5(10):970-979.
[2]Chung MJ,Kim SH,Park JW,et al. Platycodon grandiflorum root attenuates vascular endothelial cell injury by oxidized low-density lipoprotein and prevents high-fat diet-induced dyslipidemia in mice by up-regulating antioxidant proteins[J]. Nutr Res,2012,32(5):365-373.
[3]Brunner H,Cockcroft JR,Deanfield J,et al. Endothelial function and dysfunction. Part II:Association with cardiovascular risk factors and diseases. A statement by the Working Group on Endothelins and Endothelial Factors of the European Society of Hypertension[J]. J Hypertens,2005,23(2):233-246.
[4]Chen M,Li W,Zhang Y,et al. MicroRNA-20a protects human aortic endothelial cells from Ox-LDL-induced inflammation through targeting TLR4 and TXNIP signaling[J]. Biomed Pharmacother,2018,103(4):191-197.
[5]Fang F,Yang Y,Yuau Z,et al. Myocardin-related transcription factor a mediates ox LDL-induced endothelial injury[J]. Circ Res,2011,108(6):797-807.
[6]Endemann DH,Schiffrin EL. Endothelial dysfunction[J].J Am Soc Nephrol,2004,15(8):1983-1992.
[7]Hulsmans M,Holvoet P. MicroRNA-containing microvesicles regulating inflammation in association with atherosclerotic disease[J]. Cardiovasc Res,2013,100(1):7-18.
[8]Schober A,Schober A,Nazarijahantigh M,et al. MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1[J]. Nat Med,2015,20(4):368-376.
[9]Zhang X,Mao H,Chen JY,et al. Increased expression of microRNA-221 inhibits PAK1 in endothelial progenitor cells and impairs its function via c-Raf/MEK/ERK pathway[J]. Biochem Biophys Res Commun,2013,431(3):404-408.
[10]Zhang L,Zhou M,Qin G,et al. MiR-92a regulates viability and angiogenesis of endothelial cells under oxidative stress[J]. Biochem Biophys Res Commun,2014,446(4):952-958.
[11]吴小苑,范文冬. MicroRNA-195参与切应力对血管内皮细胞炎症调控作用的研究[J].中国现代医学杂志,2018,28(10):26-31.
[12]杨鹏,罗雪兰,莫国君,等. miR-24对人脐静脉内皮细胞增殖、转移及自噬的影响[J].山东医药,2017,57(13):24-27.
[13]Pradeep AR,Suke DK,Prasad MV,et al. Expression of key executioner of apoptosis caspase-3 in periodontal health and disease[J]. J Investig Clin Dent,2016,7(2):174-179.
[14]范宗静,吴旸,唐杰,等.缺血再灌注损伤人心脏微血管内皮细胞凋亡基因Bcl-2、Bax的表达及黄芪多糖干预研究[J].中华中医药杂志,2017,32(12):5603-5606.
[15]Fukai,T. Extracellular superoxide dismutase and cardiovascular disease[J]. Cardiovasc Res,2002,55(2):239-249.
[16]Tian JN,Shi XD,Wang XK,et al. Astemizole protects against human umbilical vein endothelial cell injury induced by hydrogen peroxide via the p53 signaling pathway[J]. Mol Med Rep,2017,15(6):4286-4290.
[17]刘朝阳.黄芪注射液对老年高血压患者血小板参数及氧化应激指标的影响[J].河南中医,2015,35(8):1994-1996.
[18]Bian H,Lin JZ,Li C,et al. Myocardin-related transcription factor A(MRTFA)regulates the fate of bone marrow mesenchymal stem cells and its absence in mice leads to osteopenia[J]. Mol Metab,2016,5(10):970-979.