Effect of knocking down of cortactin expression on invasion and migration of glioma cells

Abstract

Objective To investigate the impact of Cortactin knocking down by specific siRNA on the invasion and migration of human malignant glioma cells.Methods Immunohistochemistry was employed to detect the expression of Cortactin in normal brain tissues and glioblastoma tissues,which were collected from patients performed epilepsy surgery and glioma removal surgery,respectively.Human glioma cell line U251 was routinely cultured in vitro,and divided into three groups:Cortactin-siRNA group (Cortactin-siRNA being transfected into human glioma cells U251 mediated by oligofectamine),siRNA-negative control group and blank control group (Lipofetmiane 2000); 48 h after that,Western blotting and immunofluorescent staining were used to evaluate the alteration of expression and distribution of Cortactin in the three groups.Transwell invasion assay and wound-healing assay were used to determine the invasion and migration of the cells in the three groups.Results Immunohistochemistry showed that Cortactin expressed in both normal brain tissues and glioblastoma tissues,but the expression in glioblastoma tissues was significantly increased as compared with that in normal brain tissues.Western blotting indicated that Cortactin in U251 cells was knocked down dramatically after transfecting Cortactin-siRNA,and Cortactin expression was significantly lower than that in the siRNA-negative control group and blank control group (P＜0.05).Immunofluorescence showed that Cortactin located in the cytoplasm,especially closed to the cell protruded membrane.Wound-healing assay showed that the number of migrating cells was 18.17±4.07,94.00±4.33 and 92.67±5.24 in the Cortactin-siRNA group,blank control group and siRNA-negative control group,respectively; and Tmnswell invasion assay showed that the number of invaded cells was 31.25±2.98,125.00±4.57 and 127.00±3.56,with significant differences (P＜0.05); Cortactin-siRNA group had significantly weaker invasion and migration abilities as compared with blank control group and siRNA-negative control group (P＜0.05).Conclusions The Cortactin expressions in the glioblastoma tissues are significantly increased as compared with those in the normal brain tissues.The knocking down of Cortactin expression in glioma cells reduces cells migration and invasion ability,suggesting that Cortactin might be a good candidate for anti-glioma strategy. Key words: Glioma; Cortactin; RNA interference; Migratin and invasion