PTEN磷酸酶活性对乳腺癌细胞ZR-75-1迁移能力的影响

目的:探求PTEN蛋白的磷酸酶活性对乳腺癌细胞ZR-75-1转移能力的影响。方法:采用脂质体介导法分别将野生型PTEN质粒(wt-PTEN)、磷酸酶失活的PTEN质粒(G129R-PTEN)和只具有蛋白磷酸酶活性的PTEN质粒(G129E-PTEN)转染PTEN基因缺失的人乳腺癌细胞株ZR-75-1,Western印迹法检测PTEN蛋白及P397-FAK的表达水平,体外细胞划痕实验观察PTEN磷酸酶活性对ZR-75-1细胞迁移能力的影响,细胞基质黏附试验和人工重组基底膜侵袭试验测定PTEN质粒转染和未转染的ZR-75-1细胞的黏附抑制率和侵袭抑制率,免疫组化法检测MMP-2的水平。结果:wt-PTEN、G129R-PTEN及G129E-PTEN3种质粒均成功转染ZR-75-1细胞并有PTEN蛋白的表达,其中wt-PTEN、G129E-PTEN均能抑制ZR-75-1细胞迁移;wt-PTEN和G129E-PTEN转染细胞之间的黏附抑制率和侵袭抑制率或侵袭细胞相对数均无显著性差异,但与G129R-PTEN转染的和未经转染的ZR-75-1细胞相比有显著性差异(P<0.01)。wt-PTEN和G129E-PTEN质粒转染的ZR-75-1细胞其P397-FAK水平均显著低于G129R-PTEN质粒转染的ZR-75-1细胞;wt-PTEN与G129E-PTEN质粒转染的ZR-75-1细胞MMP-2水平对比于G129R-PTEN质粒转染的和未经质粒转染的ZR-75-1细胞有显著性差异(P<0.01)。结论:具有双特异磷酸酶活性的野生型PTEN基因和只具蛋白磷酸酶活性的PTEN基因均能抑制乳腺癌细胞ZR-75-1的迁移,而磷酸酶失活的PTEN基因则无此作用。

Effect of phosphatase activity of PTEN on migration ability of human breast cancer cell line ZR-75-1

Objective: This study was to explore the effects of phosphatase activity of PTEN(phosphatase and tensin homolog deleted on chromosome 10) on migration ability of human breast cancer ZR-75-1 cells.Methods: Three kinds of plasmids,wt-PTEN plasmid,G129R-PTEN plasmid,and G129E-PTEN plasmid,were constructed and transfected into human breast cancer cell line ZR-75-1,with a deletion of PTEN gene,via liposome mediation.The expression of PTEN protein as well as the Tyr397 in phospho-focal adhesion kinase(P397-FAK) was measured by Western blotting.The effect of phosphatase activity of PTEN was observed in a cell scratch wound model in vitro.The inhibitory effects of phosphatase activity of PTEN were measured by using cell-matrix adhesion test and reconstituted basement membrane invasion technique.The expression level of MMP-2 was determined by immunohistochemical staining.Results: Three kinds of plasmids(wt-PTEN,G129R-PTEN,and G129E-PTEN) were successfully transfected into ZR-75-1 cells,respectively.All the three stably transfected cells had PTEN protein expression.Transfection of wt-PTEN and G129R-PTEN inhibited the migration of ZR-75-1 cells.There was significant difference in the inhibitory degree of cell adhesion and invasion between wtPTEN or G129E-PTEN groups and G129R-PTEN transfected or non transfected groups(P<0.01).The level of P397-FAK was significantly lower in wt-PTEN and G129E-PTEN groups compared with G129R-PTEN transfected groups.There was significant difference in the expression level of MMP-2 between G129R-PTEN-transfected and non transfected groups(P<0.01).Conclusion: Both wild-type PTEN with dual specific phosphatase activities and G129E-PTEN with only protein phosphatase activity have inhibitory effects on migration of human breast cancer cell line ZR-75-1.However G129R-PTEN without phosphatase activity have no effects.