敲低皮层肌动蛋白的表达下降胶质瘤细胞迁移和侵袭能力

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

目的探讨运用特异性干扰RNA抑制皮层肌动蛋白表达后对人胶质瘤细胞迁移、侵袭能力的影响。方法免疫组化染色检测天津医科大学总医院神经外科癫痫及胶质瘤手术治疗患者的正常脑组织及胶质母细胞瘤标本中皮层肌动蛋白的表达：体外常规培养人恶性胶质瘤细胞系U251并分为RNA干扰组、阴性对照组、空白对照组，分别转染干扰RNA序列、阴性对照序列及等量Lipofetmiane2000。48h后Westernblotting、免疫荧光染色检测3组细胞皮层肌动蛋白的表达：细胞划痕实验和Transwell细胞侵袭实验评价细胞迁移、侵袭能力的高低。结果免疫组化染色检测显示皮层肌动蛋白在正常脑组织呈弱阳性表达。在胶质母细胞瘤为强阳性表达：Westernblotting检测显示RNA干扰组细胞皮层肌动蛋白表达低于空白对照组与阴性对照组，差异有统计学意义（P〈0.05）；免疫荧光染色显示皮层肌动蛋白表达于细胞质特别是与细胞伪足密切相关的细胞膜周围。划痕实验结果显示RNA干扰组、空白对照组、阴性对照组迁移细胞数分别为18．17±4．07、94．00±4．33、92．67±5．24；Transwell细胞侵袭实验显示侵袭细胞数分别为31．25±2．98、125．00±4．57、127．00±3．56；与空白对照组与阴性对照组比较，RNA干扰组迁移、侵袭细胞数均减少，差异有统计学意义（P〈0．05）。结论皮层肌动蛋白在胶质母细胞瘤组织中的表达高于正常脑组织。敲低U251细胞皮层肌动蛋白的表达后细胞的迁移、侵袭能力下降，提示皮层肌动蛋白可作为脑胶质瘤治疗的一个靶点。

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 ceils was knocked downdramatically 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 Transwell 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.