Department of Neurosurgery, Tianjin Medical University General Hospital Laboratory of Neurooncology, Tianjin Neurological Institute, 154 Anshan Road, Tianjin 300052, ChinarnObjective To investigate the inhibitory effect of Arp2/3 complex silenced by siRNA on lamellipodia and motility capability of human glioma cells in vitro. Methods The Arp3-siRNA and non-sipencing SiRN A were transfected into human glioma cells U251 using oligofectamine, respectively. Human glioma cells were then divided into siRNA siR-NA- negative control treated group (siRNA-NC) and non-silencing siRNA treated groups (siRKA-N). RT-PCR and western blotting assay were used to detect the mRNA and protein expression of Arp3. Immunofluorescence was employed to examine the location of Arp2/3 complex in glioma cells and the morphological changes of glioma cells. Transwell invasion assay and the wound-healing assay were used to determine the invasion and migration of U251 glioma cells. Results The mRNA expression levels of Arp3 in siRNA, siRNA-NC and siRNA-N groups were 19.33% ± 2.08%, 97.33% ± 2.38%, and 96.67% ± 2.52%, respectively (P<0.01). The protein expression levels in siRNA, siRNA-NC and siRNA-N groups were 20.00% ± 3.00%, 85.33% ± 3.21%, and 84.33% ± 4.04%, respectively (P<0.01). The mRNA and protein expression levels of Arp3 in Arp3-siRNA treated U251 cells was significantly inhibited up to 80% and 76% compared with thatrnin siRNA-N treated group. The lamellipodia became small or disappeared in siRNA treated U251 glioma cells whereas were kept normal in siRNA-N treated group and siRNA-NC treated group. The invasiveness and migration capability of glioma cells in the Arp3-siRNA treated group were decreased up to 69% (P<0.01) and 68% (P<0.01) compared with the siRNA-N treated group. Conclusions The Arp2/3 complex plays an important role in the spread of glioma cells through the lamellipodia, which suggests that Arp2/3 complex might be a good candidate for anticancer strategy to prevent the invasion and migration of glioma cells.%目的 研究RNA干扰技术沉默Arp2/3复合物后对胶质瘤细胞片状伪足以及运动能力的影响.方法 脂质体介导Arp3-siRNA转染体外常规培养的U251胶质瘤细胞,同时设siRNA-NC(阴性对照组,n=3)组和siRNA-N(空白对照组,n=3)组.RT-PCR和western blot方法验证三组中Arp3 mRNA和蛋白的表达情况.免疫荧光检测不同处理组的Arp2/3复合物定位以及细胞形态变化.Transwell细胞侵袭实验和划痕实验检测三组细胞的运动能力.结果 三组细胞的Arp3 mRNA相对表达水平分别为:19.33％±2.08％、97.33％±2.38％和96.67％±2.52％(P＜0.01)；三组细胞蛋白相对表达量分别为20.00％±3.00％、85.33％±3.21％和84.33％±4.04％ (P＜0.01).与siRNA-N组比较,siRNA-Arp3组胶质瘤细胞Arp3 mRNA和蛋白的表达水平下调分别达80％和76％.免疫荧光结果显示siRNA-Arp3组细胞较siRNA-NC组和siRNA-N组细胞片状伪足明显变小甚至消失.Transwell细胞侵袭实验和划痕实验显示siRNA-Arp3组胶质瘤细胞运动能力较siRNA-N组分别下降达69％ (P＜ 0.01)和68％(P＜0.01).结论 Arp2/3复合物通过影响片状伪足的形成,从而在胶质瘤细胞的运动过程中发挥重要作用,提示Arp2/3复合物可作为治疗脑胶质瘤的一个候选靶点.
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