Rac1影响下皮层肌动蛋白对U251胶质瘤细胞迁移和侵袭的调节

Regulation of cortactin with Racl activation in migration and invasion of U251 glioma cells

目的探讨皮层肌动蛋白对胶质瘤细胞迁移、侵袭的影响及RacI在此过程中的作用。方法体外常规培养人胶质瘤U251细胞，免疫荧光染色检测细胞中皮层肌动蛋白与Racl的表达和分布；将U251细胞分为siRNA干扰组、阴性对照组、空白对照组、siRNA干扰＋RacI抑制剂组．分别转染皮层肌动蛋白siRNA序列、阴性对照序列、等量Lipofetmiane2000及皮层肌动蛋白siRNA序列＋Racl抑制剂。48h后采用Westernblotting检测4组细胞皮层肌动蛋白、Racl蛋白的表达：细胞划痕实验、Transwell细胞侵袭实验分别检测细胞的迁移、侵袭能力；免疫荧光染色检测细胞片状伪足的形成。结果免疫荧光染色检测显示U251细胞中皮层肌动蛋白与Racl在肌动蛋白聚合位点共定位；Westernblotting检测显示与阴性对照组、空白对照组比较，siRNA干扰组、siRNA干扰＋Racl抑制剂组细胞皮层肌动蛋白、Racl蛋白的表达降低，且siRNA干扰＋Racl抑制剂组细胞皮层肌动蛋白、Racl蛋白的表达低于siRNA干扰组，差异均有统计学意义（P〈0．05）；划痕实验和细胞侵袭实验显示与空白对照组与阴性对照组比较，siRNA干扰组、siRNA干扰＋Racl抑制剂组创面愈合率、穿膜细胞数减少，且siRNA干扰＋RacI抑制剂组创面愈合率、穿膜细胞数低于siRNA干扰组，差异有统计学意义（P〈0．05）；免疫荧光染色检测显示siRNA干扰组、siRNA干扰＋Rac1抑制剂组细胞的片状伪足较空白对照组与阴性对照组减小，且siRNA干扰＋Rac1抑制剂组细胞的片状伪足较siRNA干扰组进一步减小。结论Rac1可能通过调节皮层肌动蛋白进而影响细胞片状伪足的大小来促进胶质瘤细胞的迁移和侵袭。联合抑制Rac1和皮层肌动蛋白可能是治疗神经胶质瘤的有效手段。

Objective To investigate the role of cortactin in migration and invasion ofU251 glioma cells and role of Racl activation in this process. Methods Human glioma U251 cells were cultured in vitro. The expressions and distributions of Racl and cortactin in U251 glioma cells were detected by immunofluorescence. U251 glioma cells assigned into 4 treatment groups： siRNA-cortactin group （transfected by siRNA specific cortactin）, siRNA-NC group （transfected by negative control RNA sequence）, siRNA-N group （transfected by empty vector） and siRNA-cortactin＋Racl group （transfected by siRNA specific cortactin and Racl inhibitor）, Forty-eighty h after grouping and each treatment, theprotein expressions of cortactin and Racl in the 4 groups were detected by Western blotting; the migration and invasion of glioma cells were evaluated by wound-healing and Transwell-chamber invasion assays; the lamellipodia of glioma cells was observed by immunofluorescence. Results Cortactin and Racl were co-localized in the front of glioma cells, where actin was polymerized and lamellipodia was formed. As compared with siRNA-NC group and siRNA-N group, siRNA-cortactin group and siRNA-cortactin＋Racl group had significantly lower cortactin and Racl expressions （P〈0.05）; siRNA-cortactin＋Racl group had significantly lower cortactin and Racl expressions as compared with siRNA-cortactin group （P〈0.05）. As compared with siRNA-NC group and siRNA-N group, siRNA-cortactin group and siRNA-cortactin＋Racl group had significantly smaller healing areas and number of perforator cells （P〈0.05）; siRNA-cortactin＋Rac 1 group had significantly smaller healing areas and number of perforator cells as compared with siRNA-cortactin group （P〈0.05）. As compared with siRNA-NC group and siRNA-N group, siRNA-cortactin group and siRNA-cortactin＋Racl group had decreased lamellipodia of glioma cells; siRNA-cortactin＋Racl group had decreased lamellipodia of glioma cells as compared with siRNA-cortactin group. Conclusion Cortactin can promote the migration and invasion of glioma cells by regulating lamellipodia formation; combined inhibition of Rac 1 and cortactin may be an effective mean for treatment ofglioma.