siRNA抑制Sema4C基因表达对人乳腺癌细胞恶性行为的影响

The Effect of Specific Inhibition of Semaphorin4C (Sema4C) Gene Expression by Small Interfering RNA on the Malignant Behavior of Human Breast Cancer Cell Line MDA-MB-231

目的:研究小干扰RNA(small interfering RNA,siRNA)抑制轴突导向蛋白分子(Semaphorin4C,Sema4C)基因的表达对人乳腺癌细胞MDA-MB-231体外迁移、侵袭及增殖的影响,并初步探讨其作用机制。方法:根据Sema4C基因设计序列特异性的siRNA(Serna4C-siRNA),在脂质体介导下转染MDA-MB-231细胞,Western blot方法检测基因封闭效应,利用细胞划痕实验、Transwell小室侵袭实验及CFSE流式细胞仪方法检测细胞转染前后迁移、侵袭及增殖能力的变化,Western blot检测磷酸化AKT(p-AKT)在转染前后细胞中表达的变化。结果:转染Sema4C-siRNA 72小时后,乳腺癌MDA-MB-231细胞株(MDA-MB-231/Si)Sema4C蛋白表达明显下降,与未转染细胞MDA-MB-231相比,MDA-MB-231/Si细胞体外迁移能力减弱,侵袭及增殖能力明显下降;p-AKT表达水平在MDA-MB-231/Si细胞中明显降低。结论:Sema4C-SiRNA转染人乳腺癌MDA-MB-231细胞可下调细胞中Sema4c蛋白表达水平,Sema4C-siRNA对MDA-MB-231细胞的体外迁移、侵袭和增殖有抑制作用。

Objective： TO study the effect of specific inhibition of Semaphorin 4C （Sema4C） gene expression by small interfering RNA （siRNA） on the migration, invasion and proliferation of highly metastatic human breast cancer cell line MDA-MB-231, in vitro. Methods： The Sema4C siRNA was designed by web based software, synthesized and then transfected into MDA-MB-231 cell line by lipofectamine 2000 reagent. Western blot was used to analyze the effect of the siRNA transfection. The migration, invasion and proliferation of the Sema4C-siRNA transfected cells were assessed by monolayer wound healing assay, transwell cell invasion assay and CFSE-flow cytometry. The expression of p-AKT protein in untrans- fected ceils, negative control-transfected MDA-MB-231 cells and Sema4C-siRNA-transfected MDA-MB-231 cells were studied by Western blot analysis. Results： Significant down-regulation of the Sema4C protein was found at 72 hours after transfection of the Sema4C-siRNA. Compared with those of untransfected cells and negative control transfected cells, the migration, invasion and proliferation ability of Sema4C-siRNA-transfected MDA-MB-231 cells were notably weakened. The protein level of p-AKT was lower in the Sema4C-siRNA-transfected MDA-MB-231 cells. Conclusion： The in vitro transient transfection of synthesized Sema4C-siRNA on the MDA-MB-231 cell line can down-regulate the expression of Sema4C protein in the cells. Sema4C-siRNA plays a vital role in suppressing migration, invasion and proliferation of MDA-MB-231 cells, which may be related to PI3K-AKT cell signal pathway.