摘要
目的:观察抑制恶性胶质瘤LN229细胞中Girdin蛋白的表达对细胞运动的影响,并探讨其分子机制。方法:用小RNA干扰技术降低恶性胶质瘤细胞株LN229中Girdin蛋白的表达水平,通过Western blotting技术验证其降表达效率,并筛选出稳定降表达的克隆命名为siGirdin/LN229,阴性对照组命名为scr/LN229。利用增殖实验检测LN229细胞的存活率;应用划痕实验和侵袭实验检测细胞的运动变化。通过慢病毒感染技术来提高Girdin在LN229细胞中的表达水平,使用嘌呤霉素筛选2周建立稳定的细胞系,并用Western blot验证Girdin表达水平。结果:Western blotting验证小RNA干扰技术有效沉默了LN229细胞中Girdin的表达。增殖实验显示siGirdin/LN229细胞增殖能力下降(P<0.05),至第6天时实验组细胞数为(19.98±2.54)×104;对照组细胞数为(61.04±5.26)×104。划痕实验发现细胞非定向运动能力减弱(P<0.05),24 h时实验组细胞运动的距离为(21.83±2.52)μm;对照组为(44.16±2.89)μm。Western blotting结果表明LN229细胞中Girdin的表达增高,增殖实验显示过表达Girdin对LN229细胞增殖能力无影响。体外细胞侵袭实验中实验组穿过Matrigel胶的细胞数为(14.67±2.08)个;对照组为(32.67±3.06)个,结果显示Girdin降表达后细胞的侵袭能力较对照组细胞显著下降(P<0.05)。结论:Girdin和胶质瘤细胞增殖和运动能力密切相关,Girdin蛋白表达降低可抑制LN229细胞的增殖、迁移和侵袭能力。
Objective： The objectives of this study were to investigate the role of Girdin protein in the motility ofLN229 glioblastoma cells using small RNA interference technology and to determine the underlying molecular mechanisms involved. Methods： The expression of Girdin was knocked down in LN229 glioblastoma cells by Girdin siRNA. It was identified by Western blot analysis. Stable clones that could detect the cell survival ratio were used in the proliferation assay. Scratch and invasion in vitro assays were also used to investigate the motility of the cells. Results： Western blot analysis showed that RNA interference significantly decreased the expression level of Girdin. Proliferation assay revealed that the munber of siGirdin/LN229 cells decreased compared with that of scr/LN229 cells. On the 6th day, the number of siGirdin/LN229 cells was 19.98 ± 2.54 × 10^4, whereas that of scr/LN229 cells was 61.04± 5.26×10^4. At 24 h after wounding, the migrated distances of the siGirdin/LN229 and scr/LN229 cells were 21.83 ± 2.52 and 44.16 ± 2.89 μm, respectively. Moreover, although the expression level of Girdin increased, the number of PCDH-p-Girdin/LN229 cells did not increase compared with that of PCDH-p-vector/ LN229 cells. Invasion in vitro assay demonstrated that the numbers of siGirdin/LN229 and scr/LN229 transmembrane cells were 14.67 ± 2.08 and 32.67 ± 3.06, respectively. Conclusion： Girdin is essential to the proliferation and motility of glioblastoma cells.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2013年第1期8-11,共4页
Chinese Journal of Clinical Oncology
