GTP酶激活蛋白(Src同源结构域3)结合蛋白1在乳腺癌细胞MDA-MB-231体外迁移中的作用

Function of Ras-GTPase activating protein(Src homology domain 3) binding protein 1 in the in vitro migration of human breast cancer cell MDA-MB-231

目的:研究GTP酶激活蛋白(Src同源结构域3)结合蛋白1[Ras-GTPase activating protein(Src homology do-main 3)binding protein 1,G3BP1]在乳腺癌细胞MDA-MB-231体外迁移过程中的作用。方法:人乳腺癌细胞MDA-MB-231培养于RPMI1640培养基中;Western blot检测无表皮生长因子(Epidermal growth factor,EGF)刺激,以及10 ng/ml EGF分别刺激30秒、1分钟、2分钟和5分钟条件下乳腺癌细胞内G3BP1、蛋白激酶Cζ(Protein kinase Cζ,PKCζ)、Akt、pPKCζThr410/403及pAktSer473的表达水平;免疫共沉淀法检测乳腺癌细胞内G3BP1与PKCζ的相互作用;转染小干扰RNA抑制内源性G3BP1的表达;趋化实验和侵袭实验分别检测G3BP1抑制前后MDA-MB-231细胞穿过10μm聚碳酸酯膜及人工基底膜的细胞数。结果:Western blot检测显示G3BP1在MDA-MB-231细胞内过表达;无EGF刺激时MDA-MB-231细胞内G3BP1,PKCζ与Akt均过表达,pPKCζThr410/403及pAktSer473不表达或低于Western blot检测下限;10 ng/ml EGF刺激后G3BP1、PKCζ与Akt表达水平不变,pPKCζThr410/403及pAktSer473的表达刺激时间增加而升高,至5分钟时达到峰值;在乳腺癌细胞内G3BP1与PKCζ具有相互作用;G3BP1表达抑制后乳腺癌细胞的趋化能力和侵袭能力均显著下降(P<0.05,P<0.05)。结论:G3BP1通过与PKCζ相互作用,在乳腺癌细胞MDA-MB-231的体外迁移过程中发挥重要作用。

Objective：To study the function of Ras-GTPase activating protein（Src homology domain 3） binding protein 1（G3BP1） in the in vitro migration of breast cancer cell MDA-MB-231.Methods： Human breast cancer cell MDA-MB-231 was cultured in medium RPMI1640.Expression of G3BP1,PKCζ,Akt,pPKCζThr410/403 and pAktSer473 without EGF stimulation,10 ng/ml EGF stimulation for 30 seconds,1 minute,2 minutes and 5 minutes were detected by Western blot.Interaction of G3BP1 and PKCζ was detected by Co-Immunoprecipitation.Intracellular G3BP1 expression was blocked by transfection of siRNA.Boyden Chamber Assay and Invasion Assay were separately performed to detect MDA-MB-231＇s chemotaxis and invasion ability before and after G3BP1 block.Results： Western blot showed an overexpression of G3BP1 in MDA-MB-231.Without EGF stimulation,there was an overexpression of G3BP1,PKCζ and Akt,and no expression of pPKCζThr410/403 and pAktSer473.After 10 ng/ml EGF stimulation,there was no change of the expression of G3BP1,PKCζ and Akt,and an increase of the expression of pPKCζThr410/403 and pAktSer473 which reached the peak after 5 minutes＇ treatment.G3BP1 and PKCζ interacts with eachother.There were great decrease in the number of cells that passed 10 μm polycarbonate membrane and matrigel after G3BP1 expression block（P0.05,P0.05）.Conclusion： Through interaction with PKCζ,G3BP1 exerts an important function in the in vitro migration of cancer cell MDA-MB-231.