siRNA抑制VEGF基因表达对鼻咽癌细胞生物学行为的影响

Suppression of VEGF via RNA interference modulates the biological features of human nasopharyngeal carcinoma CNE-2 cell line

目的:应用RNA干扰(RNAi)技术抑制人鼻咽低分化鳞状上皮细胞癌细胞株CNE-2中血管内皮生长因子(VEGF)的表达,研究VEGF对鼻咽癌细胞生物学行为的影响。方法:构建针对VEGF的siRNA真核表达载体pU-VEGF-siR-NA,将其表达载体经脂质体LipofectamineTM2000转染至CNE-2细胞,荧光显微镜下观察转染效率,采用RT-PCR、Western blot方法检测转染后CNE-2细胞中VEGF mRNA和蛋白的表达。通过流式细胞仪分析细胞周期的分布,并应用平板克隆形成实验、Transwell侵袭小室模型观察转染后CNE-2细胞恶性生物学行为的变化。结果:pU-VEGF-siRNA转染后CNE-2细胞株中VEGF mRNA和蛋白表达较pU-siCONT组、空白对照组显著下降(P<0.05),细胞周期被阻滞在G1期,细胞生长缓慢,体外侵袭能力下降。结论:通过RNAi技术阻断VEGF的表达,可抑制CNE-2细胞的生长、增殖、迁徙,提示VEGF在鼻咽癌的发生、发展过程中起重要作用。

Objective：To study the effect of vascular endothelial growth factor （VEGF） down-regulation by small interfering RNA （RNAi） on the biological features of nasopharyngeal carcinoma cell line CNE-2. Methods ： CNE-2 cells were transfected with synthetic small interfering RNA （siRNA） targeting VEGF. Transfection efficiencies were monitored by fluorescent microscopy. Expression of VEGF mRNA and protein were respectively measured by reverse transcription polymerase chain reaction （RT-PCR） and Western blot. The distribution of cell cycle phases was determined using flow cytometry. The proliferative and invasive ability of CNE-2 cells was evaluated by the colony-forming unit assay and transwell migration assay, respectively. Results：Both VEGF mRNA and protein expres- sion were significantly decreased in the experimental group compared with the control group （ P 〈 0. 05 ）. The cell cycle was arrested in the G1 phase. The proliferation and invasiveness of treated CNE-2 cells were inhibited in vitro. Conclusion： Delivery of siRNA targeting VEGF seems efficient in down-regulating VEGF expression and diminishing the growth, proliferation and invasiveness of CNE-2 cells, suggesting that siRNA-based strategy targeting VEGF may lay a foundation for the clinical management of nasopharyngeal carcinoma.