siRNA特异性沉默septin2基因抑制大肠癌LoVo细胞迁移

Inhibitory effects of septin 2 gene silencing induced by siRNA on migration of human colorectal cancer cell line LoVo

目的:检测新型骨架蛋白septin 2在不同转移能力的人结直肠癌细胞株中的表达水平,探究其在结直肠癌细胞迁移中的作用。方法:采用实时荧光定量RT-PCR及Western blotting检测septin 2基因及其蛋白在高转移潜能细胞株LoVo和低转移潜能癌细胞株HT-29、SW480、HCT-116中的表达;用siRNA干扰方法沉默高表达的septin 2基因,实时荧光定量RT-PCR检测干扰效率,免疫荧光共聚焦实验观测干扰前后septin 2蛋白表达的变化,划痕实验检测干扰前后细胞的迁移能力。结果:septin 2在LoVo细胞的表达水平显著高于HT-29、SW480及HCT-116细胞(P<0.05)。沉默septin 2基因后,LoVo细胞septin 2 mRNA表达降低(P<0.05);septin 2与纤维状肌动蛋白(F-actin)存在共表达;F-actin表达减弱,应力纤维减少,片状伪足细而短;细胞迁移能力减弱。结论:septin 2基因在LoVo细胞高表达,干扰其表达能导致细胞骨架重构,从而降低细胞迁移能力。

AIM： To investigate the expression of septin 2 in human colorectal cancer cell lines and the effect of septin 2 on the capacity of migration of human colorectal cancer cell line LoVo. METHODS ： Real - time fluorescence quantitative RT - PCR and Western blotting analysis were used to determine the mRNA and protein levels of septin 2 in dif- ferent metastatic potential cell lines, respectively. The expression of septin 2 in I^Vo cells was silenced by siRNA. The ef- ficacy of siRNA was confirmed by real - time fluorescence quantitative RT - PCR. Septin 2 and its co - localization with F -actin were measured by immunofluorescence method. The migration of transfected cells was analyzed by scratch test. RESULTS ： The expression of septin 2 in LoVo cells was significantly higher than that in low metastatic potential cell lines such as SW480, HCT - 116 and HT - 29 at both mRNA and protein levels. The mRNA expression of septin 2 was success- fully silenced in LoVo cells by siRNA. Cell wound closure rate was also decreased in septin 2 siRNA group as compared with control group （ both P 〈 0. 05 ）. The co - expression of septin 2 and F - actin formed the typical filamentous - granular organization, and down - regulation of septin 2 resulted in cell skeleton disturbance with less or shorter pseudopodium and decreased stress fibers. CONCLUSION： Septin 2 is highly expressed in LoVo cells. Partial deletion of septin 2 represses the capability of tumor cell migration.