Ox-LDL对THP-1巨噬细胞迁移功能、microRNA21表达及MAPK通道磷酸化的影响
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摘要
目的:探讨氧化型低密度脂蛋白(ox-LDL)对THP-1巨噬细胞迁移功能、microRNA21(miR-21)表达及丝裂原活化蛋白激酶(MAPK)通路的影响。方法:以160nmol/L佛波酯(PMA)诱导THP-1细胞分化成巨噬细胞。根据是否采用ox-LDL处理及脂质体介导miR-21抑制剂转染THP-1巨噬细胞,分为空白对照组(未进行oxLDL处理及脂质体转染),ox-LDL组(采用50mg/L ox-LDL处理,未进行脂质体转染),miR-21抑制剂组(脂质体介导miR-21抑制剂转染后采用50mg/L ox-LDL处理),miR-21抑制剂阴性对照组(脂质体介导miR-21抑制剂阴性对照转染后采用50mg/L ox-LDL处理)。Transwell法检测THP-1巨噬细胞迁移数,实时定量PCR检测miR-21表达情况,Western-blot法检测双特异性磷酸-8(DUSP-8)表达水平及磷酸化水平。结果:与空白对照组比较,ox-LDL组THP-1巨噬细胞迁移数显著增加[(74.10±15.10)比(184.10±26.28)],miR-21表达水平[(1.00±0.21)比(2.02±0.27)],MAPK通路的JNK、P38蛋白磷酸化水平显著升高,DUSP-8蛋白表达水平降低,P均<0.01;与ox-LDL组比较,miR-21抑制剂组THP-1巨噬细胞迁移数[(184.10±26.28)比(58.50±10.24)]显著减少,miR-21表达水平[(2.02±0.27)比(0.66±0.16)],JNK、P38蛋白磷酸化水平显著降低,DUSP-8蛋白表达水平显著升高,P均<0.01。结论:ox-LDL增强巨噬细胞迁移能力,可能与其上调miR-21表达、增强MAPK通路的JNK、P38蛋白磷酸化、降低DUSP-8表达有关。
Objective:To explore influence of oxidized low density lipoprotein(ox-LDL)on migration function of THP-1 macrophages,expression of microRNA 21(miR-21)and mitogen-activated protein kinase(MAPK)pathway.Methods:Phorbol myristate acetate(PMA)of 160 nmol/L was used to induce THP-1 cells to differentiate into macrophages.According to application of ox-LDL treatment and liposomes-mediated miR-21 inhibitor transfecting THP-1 macrophages(transfection for short)or not,THP-1 macrophages were divided into blank control group(received neither ox-LDL treatment nor transfection),ox-LDL group(received 50 mg/L ox-LDL treatment without transfection),miR-21 inhibitor group(received 50 mg/L ox-LDL treatment after transfection)and miR-21 inhibitor negative-control group(received 50 mg/L ox-LDL treatment after negative-control transfection).THP-1 macrophage migration number was measured by transwell method,miR-21 expression was measured by real-time quantitative PCR,and expression of dual specific phosphate 8(DUSP-8)and phosphorylation level of MAPK pathway were measured by Western-blot method.Results:Compared with blank control group,there were significant rise in migration number of THP-1 macrophages[(74.10±15.10)vs.(184.10±26.28)],miR-21 expression[(1.00±0.21)vs.(2.02±0.27)]and phosphorylation levels of JNK and P38 protein,and significant reduction in expression of DUSP-8 protein in ox-LDL group,P<0.01 all.Compared with ox-LDL group,there were significant reductions in migration number of THP-1 macrophages[(184.10±26.28)vs.(58.50±10.24)],miR-21 expression [(2.02±0.27)vs.(0.66±0.16)]and phosphorylation levels of JNK and P38 protein,and significant rise in expression of DUSP-8 protein in ox-LDL group,P<0.01 all.Conclusion:Ox-LDL enhances migration function of macrophages,which may be related to its effects of upregulating miR-21 expression,enhancing phosphorylation of JNK and P38 protein of MAPK pathway and reducing DUSP-8 expression.
Objective:To explore influence of oxidized low density lipoprotein(ox-LDL)on migration function of THP-1 macrophages,expression of microRNA 21(miR-21)and mitogen-activated protein kinase(MAPK)pathway.Methods:Phorbol myristate acetate(PMA)of 160 nmol/L was used to induce THP-1 cells to differentiate into macrophages.According to application of ox-LDL treatment and liposomes-mediated miR-21 inhibitor transfecting THP-1 macrophages(transfection for short)or not,THP-1 macrophages were divided into blank control group(received neither ox-LDL treatment nor transfection),ox-LDL group(received 50 mg/L ox-LDL treatment without transfection),miR-21 inhibitor group(received 50 mg/L ox-LDL treatment after transfection)and miR-21 inhibitor negative-control group(received 50 mg/L ox-LDL treatment after negative-control transfection).THP-1 macrophage migration number was measured by transwell method,miR-21 expression was measured by real-time quantitative PCR,and expression of dual specific phosphate 8(DUSP-8)and phosphorylation level of MAPK pathway were measured by Western-blot method.Results:Compared with blank control group,there were significant rise in migration number of THP-1 macrophages[(74.10±15.10)vs.(184.10±26.28)],miR-21 expression[(1.00±0.21)vs.(2.02±0.27)]and phosphorylation levels of JNK and P38 protein,and significant reduction in expression of DUSP-8 protein in ox-LDL group,P<0.01 all.Compared with ox-LDL group,there were significant reductions in migration number of THP-1 macrophages[(184.10±26.28)vs.(58.50±10.24)],miR-21 expression [(2.02±0.27)vs.(0.66±0.16)]and phosphorylation levels of JNK and P38 protein,and significant rise in expression of DUSP-8 protein in ox-LDL group,P<0.01 all.Conclusion:Ox-LDL enhances migration function of macrophages,which may be related to its effects of upregulating miR-21 expression,enhancing phosphorylation of JNK and P38 protein of MAPK pathway and reducing DUSP-8 expression.
引文
[1]Ross R.The pathogenesis of atherosclerosis:aperspective for the 1990s[J].Nature,1993,362(6423):801-809.
[2]De Boer OJ,van der Wal AC,Becker AE.Atherosclerosis,inflammation,and infection[J].J Pathol,2000,190(3):237-243.
[3]Lam MC,Tan KC,Lam KS.Glycoxidized low-density lipoprotein regulates the expression of scavenger receptors in THP-1macrophages[J].Atherosclerosis,2004,177(2):313-320.
[4]Chen T,Huang Z,Wang L,et al.MicroRNA-125a-5p partly regulates the inflammatory response,lipid uptake,and ORP9expression in oxLDL-stimulated monocyte/macrophages[J].Cardiovasc Res,2009,83(1):131-139.
[5]Gao L,Wang CQ.Role of miR-21in monocyte/macrophage functions and Atherosclerosis[J].Doctor's degree thesis,Shanghai Jiao Tong University,2016.
[6]Liu S,Li W,Xu M,et al.miR-21targets DUSP8to promote collagen synthesis in high glucose treated primary cardiac fibroblasts[J].Can J Cardiol,2014,30(12):1689-1699.
[7]Ross R.Atherosclerosis an inflammatory disease[J].N Engl J Med,1999,340(2):115-126.
[8]Ghattas A,Griffiths HR,Devitt A,et al.Monocytes in coronary artery disease and atherosclerosis:where are we now?[J].J Am College of Cardiol,2013,62(17):1541-1551.
[9]Aguda BD,Kim Y,Piper-Hunter MG,et al.MicroRNA regulation of a cancer network:consequences of the feedback loops involving miR-17-92,E2F,and Myc[J].Proc Natl AcadSci USA,2008,105(50):19678-19683.
[10]Xiao C,Rajewsky K.MicroRN A control in the immune system:basic principles[J].Cell,2009,136(1):26-36.
[11]O'Hara SP,Mott JL,Splinter PL,et al.MicroRNAs:key modulators of post-transcriptional gene expression[J].Gastroenterol,2009,136(1):17-25.
[12]Yin C,Salloum FN,Kukreja RC.A novel role of microRNA in late preconditioning:upregulation of endothelial nitric oxide synthase and heat shock protein 70[J].Circ Res,2009,104(5):572-575.
[13]McDonald RA,Halliday CA,Miller AM,et al.Reducing in stent restenosis:therapeutic manipulation of miRNA in vascular remodeling and inflammation[J].J Am Coll Cardiol,2015,65(21):2314-2327.
[14]Wang Z,Brandt S,Medeiros A,et al.MicroRNA 21is a homeostatic regulator of macrophage polarization and prevents prostaglandin E2-mediated M2 generation[J].PLoS One,2015,10(2):e0115855.
[15]Ji R,Cheng Y,Yue J,et al.MicroRNA expression signature and antisense-mediated depletion reveal an essential role of MicroRNA in vascular neointimal lesion formation[J].Circulation Research,2007,100(11):1579-1588.
[16]Lin Y,Liu X,Cheng Y,et al.Involvement of MicroRNAs in hydrogen peroxide-mediated gene regulation and cellular injury response in vascular smooth muscle cells[J].J Biological Chemistry,2009,284(12):7903-7913.
[17]Oudit GY,Sun H,Kerfant BG,et al.The role of phosphoinositide-3kinase and PTEN in cardiovascular physiology and disease[J].J Mol Cellular Cardiol,2004,37(2):449-471.
[18]Chistiakov DA,Orekhov AN,Bobryshev YV.Vascular smooth muscle cell in atherosclerosis[J].Acta Physiol(Oxf),2015,214(1):33-50.
[19]Kondoh K,Nishida E.Regulation of MAP kinases by MAP kinase phosphatases[J].Biochimica Et Biophysica Acta,2007,1773(8):1227-1237.
[2]De Boer OJ,van der Wal AC,Becker AE.Atherosclerosis,inflammation,and infection[J].J Pathol,2000,190(3):237-243.
[3]Lam MC,Tan KC,Lam KS.Glycoxidized low-density lipoprotein regulates the expression of scavenger receptors in THP-1macrophages[J].Atherosclerosis,2004,177(2):313-320.
[4]Chen T,Huang Z,Wang L,et al.MicroRNA-125a-5p partly regulates the inflammatory response,lipid uptake,and ORP9expression in oxLDL-stimulated monocyte/macrophages[J].Cardiovasc Res,2009,83(1):131-139.
[5]Gao L,Wang CQ.Role of miR-21in monocyte/macrophage functions and Atherosclerosis[J].Doctor's degree thesis,Shanghai Jiao Tong University,2016.
[6]Liu S,Li W,Xu M,et al.miR-21targets DUSP8to promote collagen synthesis in high glucose treated primary cardiac fibroblasts[J].Can J Cardiol,2014,30(12):1689-1699.
[7]Ross R.Atherosclerosis an inflammatory disease[J].N Engl J Med,1999,340(2):115-126.
[8]Ghattas A,Griffiths HR,Devitt A,et al.Monocytes in coronary artery disease and atherosclerosis:where are we now?[J].J Am College of Cardiol,2013,62(17):1541-1551.
[9]Aguda BD,Kim Y,Piper-Hunter MG,et al.MicroRNA regulation of a cancer network:consequences of the feedback loops involving miR-17-92,E2F,and Myc[J].Proc Natl AcadSci USA,2008,105(50):19678-19683.
[10]Xiao C,Rajewsky K.MicroRN A control in the immune system:basic principles[J].Cell,2009,136(1):26-36.
[11]O'Hara SP,Mott JL,Splinter PL,et al.MicroRNAs:key modulators of post-transcriptional gene expression[J].Gastroenterol,2009,136(1):17-25.
[12]Yin C,Salloum FN,Kukreja RC.A novel role of microRNA in late preconditioning:upregulation of endothelial nitric oxide synthase and heat shock protein 70[J].Circ Res,2009,104(5):572-575.
[13]McDonald RA,Halliday CA,Miller AM,et al.Reducing in stent restenosis:therapeutic manipulation of miRNA in vascular remodeling and inflammation[J].J Am Coll Cardiol,2015,65(21):2314-2327.
[14]Wang Z,Brandt S,Medeiros A,et al.MicroRNA 21is a homeostatic regulator of macrophage polarization and prevents prostaglandin E2-mediated M2 generation[J].PLoS One,2015,10(2):e0115855.
[15]Ji R,Cheng Y,Yue J,et al.MicroRNA expression signature and antisense-mediated depletion reveal an essential role of MicroRNA in vascular neointimal lesion formation[J].Circulation Research,2007,100(11):1579-1588.
[16]Lin Y,Liu X,Cheng Y,et al.Involvement of MicroRNAs in hydrogen peroxide-mediated gene regulation and cellular injury response in vascular smooth muscle cells[J].J Biological Chemistry,2009,284(12):7903-7913.
[17]Oudit GY,Sun H,Kerfant BG,et al.The role of phosphoinositide-3kinase and PTEN in cardiovascular physiology and disease[J].J Mol Cellular Cardiol,2004,37(2):449-471.
[18]Chistiakov DA,Orekhov AN,Bobryshev YV.Vascular smooth muscle cell in atherosclerosis[J].Acta Physiol(Oxf),2015,214(1):33-50.
[19]Kondoh K,Nishida E.Regulation of MAP kinases by MAP kinase phosphatases[J].Biochimica Et Biophysica Acta,2007,1773(8):1227-1237.