腺苷酸环化酶相关蛋白1对人脑胶质瘤细胞迁移、侵袭和黏附的影响
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摘要
目的探讨腺苷酸环化酶相关蛋白1(CAP1)对人脑胶质瘤细胞株U87、U251迁移、侵袭和黏附的影响及其机制。方法运用小干扰RNA(siRNA)技术分别转染U87、U251细胞,Western blot检测CAP1、MMP-2、MMP-9、黏着斑激酶(FAK)和含有酪氨酸激酶活性的Src的表达。应用细胞划痕实验及Transwell迁移、侵袭实验检测U87、U251细胞的迁移和侵袭能力。应用细胞黏附实验检测细胞黏附能力。结果与阴性对照组比较,CAP1-siRNA组U87、U251细胞中CAP1蛋白表达量降低,MMP-2、MMP-9、FAK和Src的蛋白表达量均减少(P<0.05)。CAP1-siRNA组较阴性对照组细胞的迁移能力降低(P<0.05)。沉默CAP1后,U87、U251细胞的迁移、侵袭能力下降(P<0.05)。CAP1-siRNA组黏附细胞个数少于阴性对照组(P<0.05)。结论 CAP1-siRNA靶向干扰了CAP1的表达,同时下调MMP-2、MMP-9、FAK和Src的表达。下调CAP1的表达水平能够显著降低人脑胶质瘤细胞株U87、U251迁移、侵袭和黏附能力。
Objective To investigate the effect and underlying mechanism of adenylate cyclaseassociated protein 1(CAP1)on cell migration,invasion and adhesion of human brain glioma cells U87 and U251.Methods Small interfering RNA(siRNA)was used to transfect U87 and U251 cells,respectively.The expressions of CAP1 and matrix metallopeptidase-2(MMP-2),MMP-9,focal adhesion kinase(FAK)and tyrosine kinase(Src)proteins in U87 and U251 cells were detected after CAP1 was knockdown using Western blot.The abilities of U87,U251 cells migration and invasion were measured by wound-healing assay and transwell migration and invasion assays,respectively.The adhesion ability was detected by cell attachment assay.Results Compared with negative controlsiRNA group,the expression of CAP1 was decreased obviously in CAP1-siRNA group and the expression levels of MMP-2,MMP-9,FAK and Src were down-regulated(P<0.05).The migration ability of U87 and U251 cells was decreased obviously after CAP1 was knockdown(P<0.05).The abilities of migration and invasion of U87 and U251 cells in CAP1-siRNA group were decreased(P<0.05).The number of adhered cells in CAP1-siRNA group was significantly less than that in negative control-siRNA group(P<0.05).Conclusion CAP1-siRNA targetedly interferes the expressions of CAP1 and down-regulates the expressions of MMP-2,MMP-9,FAK and Src.The abilities of migration,invasion and adhesion of U87 and U251 cells can be suppressed by the downregulation of CAP1 expression.
Objective To investigate the effect and underlying mechanism of adenylate cyclaseassociated protein 1(CAP1)on cell migration,invasion and adhesion of human brain glioma cells U87 and U251.Methods Small interfering RNA(siRNA)was used to transfect U87 and U251 cells,respectively.The expressions of CAP1 and matrix metallopeptidase-2(MMP-2),MMP-9,focal adhesion kinase(FAK)and tyrosine kinase(Src)proteins in U87 and U251 cells were detected after CAP1 was knockdown using Western blot.The abilities of U87,U251 cells migration and invasion were measured by wound-healing assay and transwell migration and invasion assays,respectively.The adhesion ability was detected by cell attachment assay.Results Compared with negative controlsiRNA group,the expression of CAP1 was decreased obviously in CAP1-siRNA group and the expression levels of MMP-2,MMP-9,FAK and Src were down-regulated(P<0.05).The migration ability of U87 and U251 cells was decreased obviously after CAP1 was knockdown(P<0.05).The abilities of migration and invasion of U87 and U251 cells in CAP1-siRNA group were decreased(P<0.05).The number of adhered cells in CAP1-siRNA group was significantly less than that in negative control-siRNA group(P<0.05).Conclusion CAP1-siRNA targetedly interferes the expressions of CAP1 and down-regulates the expressions of MMP-2,MMP-9,FAK and Src.The abilities of migration,invasion and adhesion of U87 and U251 cells can be suppressed by the downregulation of CAP1 expression.
引文
[1]张雅旋.E2F2基因在胶质瘤中的表达及其对胶质瘤细胞生长与代谢的影响[J].江苏医药,2014,16(40):1861-1864.
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[9]Nishida Y,Shima F,Sen H,et al.Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway[J].J Biol Chem,1998,273(43):28019-28024.
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[12]Yamazaki K,Takamura M,Masugi Y,et al.Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility[J].Lab Invest,2009,89(4):425-432.
[13]范月超,张慧,郑骏年,等.靶向Dicer酶的小干扰RNA对人脑胶质瘤细胞生物学行为的影响[J].中华实验外科杂志,2013,30(7):1475-1477.
[14]Fan YC,Zhu YS,Mei PJ,et al.Cullin1regulates proliferation,migration and invasion of glioma cells[J].Med Oncol,2014,31(10):227.
[15]Siesser PM,Hanks SK.The signaling and biological implications of FAK overexpression in cancer[J].Clin Cancer Res,2006,12(11Pt 1):3233-3237.
[16]Summy JM,Gallick GE.Src family kinases in tumor progression and metastasis[J].Cancer Metastasis Rev,2003,22(4):337-358.
[2]Hood JD,Cheresh DA.Role of integrins in cell invasion and migration[J].Nat Rev Cancer,2002,2(2):91-100.
[3]Matviw H,Yu G,Young D.Identification of a human cDNAencoding aprotein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins[J].Mol Cell Biol,1992,12(11):5033-5040.
[4]Field J,Vojtek A,Ballester R,et al.Cloning and characterization of CAP,the S.cerevisiae gene encoding the 70kd adenylyl cyclase-associated protein[J].Cell,1990,61(2):319-327.
[5]Fedor-Chaiken M,Deschenes RJ,Broach JR.SRV2,agene required for RAS activation of adenylate cyclase in yeast[J].Cell,1990,61(2):329-340.
[6]Moriyama K,Yahara I.Human CAP1is a key factor in the recycling of cofilin and actin for rapid actin turnover[J].J Cell Sci,2002,115(Pt 8):1591-1601.
[7]Hubberstey AV,Mottillo EP.Cyclase-associated proteins:CAPacity for linking signal transduction and actin polymerization[J].FASEB J,2002,16(6):487-499.
[8]Hua M,Yan S,Deng Y,et al.CAP1is overexpressed in human epithelial ovarian cancer and promotes cell proliferation[J].Int J Mol Med,2015,35(4):941-949.
[9]Nishida Y,Shima F,Sen H,et al.Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway[J].J Biol Chem,1998,273(43):28019-28024.
[10]Freeman NL,Chen Z,Horenstein J,et al.An actin monomer binding activity localizes to the carboxyl-terminal half of the Saccharomyces cerevisiae cyclase-associated protein[J].J Biol Chem,1995,270(10):5680-5685.
[11]Makkonen M,Bertling E,Chebotareva NA,et al.Mammalian and malaria parasite cyclase-associated proteins catalyze nucleotide exchange on G-actin through a conserved mechanism[J].J Biol Chem,2013,288(2):984-994.
[12]Yamazaki K,Takamura M,Masugi Y,et al.Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility[J].Lab Invest,2009,89(4):425-432.
[13]范月超,张慧,郑骏年,等.靶向Dicer酶的小干扰RNA对人脑胶质瘤细胞生物学行为的影响[J].中华实验外科杂志,2013,30(7):1475-1477.
[14]Fan YC,Zhu YS,Mei PJ,et al.Cullin1regulates proliferation,migration and invasion of glioma cells[J].Med Oncol,2014,31(10):227.
[15]Siesser PM,Hanks SK.The signaling and biological implications of FAK overexpression in cancer[J].Clin Cancer Res,2006,12(11Pt 1):3233-3237.
[16]Summy JM,Gallick GE.Src family kinases in tumor progression and metastasis[J].Cancer Metastasis Rev,2003,22(4):337-358.