应用小分子干扰RNA技术以慢病毒为载体抑制乳腺癌淋巴管生成因子VEGF-C的实验研究

The study of down-regulating breast cancer lymphangiogenesis factor VEGF-C via lenti-virus based RNA interference technology

目的研究VEGF-C特异性小干扰RNA(small interference RNA,si RNA)对乳腺癌淋巴管生成因子-血管内皮生长因子C(vascular endothelial growth factor C,VEGF-C)表达水平及乳腺癌细胞转移能力的影响。方法实验分为感染RNAi靶点病毒的细胞组(KD组)、感染阴性对照病毒的细胞组(NC组)和未感染任何病毒的细胞组(CON组)。以慢病毒为载体将靶向VEGF-C基因的si RNA导入体外培养的乳腺癌MDA-MB-231细胞系,应用Real-time PCR和Western blot技术检测各组细胞VEGF-C m RNA水平和蛋白水平表达差异,通过小室侵袭实验观察各组细胞侵袭能力。结果与细胞体外共培养3d后慢病毒具有较高的感染效率,在荧光显微镜下观察其感染效率达70%以上。与NC组、CON组比较,KD组细胞VEGF-C在m RNA水平表达明显降低,抑制率达60%以上,差异有统计学意义(P<0.05);在蛋白水平,KD组也受到明显抑制,条带明显模糊变淡。小室侵袭实验显示,KD组较NC组、CON组侵袭能力明显降低,差异有统计学意义(P<0.05)。结论以慢病毒为载体,特异性si RNA能够有效抑制VEGF-C的表达及乳腺癌细胞的侵袭能力,为预防与治疗乳腺癌淋巴道转移提供实验依据,同时也为下一步动物实验奠定基础。

Objective To investigate the effect of si RNA on VEGF-C expression and the breast cancer cell invasion ability. Methods The human breast cancer cell MDA-MB-231 were cultured and divided into three groups： cells transfected with lenti-virus based si RNA targeting VEGF-C（KD group）, cells transfected with lenti-virus based negative si RNA（NC group）, regular cells（CON group）. VEGF-C expression was quantified by real-time polymerase chain reaction and western blot. Small well invasion test was used to compare the invasion ability among each group. Results The electronic fluorescence microscope showed that lenti-virus had a high transfection rate that more than 70%. The m RNA and protein expression of VEGF-C in the breast cancer cell line, MDA-MB-231, stably transfected with VEGF-C-si RNA lenti-virus vector, were significantly downregulated（P〈0.05）. The small well invasion test showed that the invasion ability was obviously inhibited in the KD group（P〈0.05）. Conclusion VEGF-C si RNA effectively inhibit VEGF-C protein and m RNA expression and the breast cancer cell invasion ability. These findings show a clue to prevent and cure breast cancer lymphatic metastasis, and also make a good preparation for further animal research.