Down-regulating VEGF Expression Enhances Radiosensitivity of Human Nasopharyngeal Carcinoma Cells by RNA Interference
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摘要: 目的 应用RNA干扰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）表达，研究阻断VEGF基因表达对鼻咽癌细胞放射敏感度的影响及机制。方法 构建针对VEGF的siRNA真核表达载体pU-VEGF-siRNA（CNE-2组）、1个阴性对照质粒（CNE-2/Neg-siRNA 组）、经脂质体转染至CNE-2细胞（CNE-2/VEGF-siRNA组），用平板克隆形成实验检测在6MV-X线0、2、4、6、8、10 Gy照射后克隆形成能力及通过单击多靶模型、线性二次模型拟合放射生物学参数，流式细胞检测分析细胞周期和细胞凋亡的变化，用RT-PCR定量分析三组细胞中Cyclin D1、Cyclin E、P16和P53的mRNA表达。结果 经6MV-X线0、2、4、6、8、10 Gy照射后细胞存活率显著下降，CNE-2/VEGF-siRNA组细胞经D0和2 Gy照射后的存活分数明显低于CNE-2组、CNE-2/Neg-siRNA组；CNE-2/VEGF-siRNA组中G1/S期细胞周期阻滞更为明显。在Cyclin D1、Cyclin E、p16和p53基因中，Cyclin D1mRNA表达在放疗后6、12和24 h进行性升高，差异有统计学意义，而其他基因变化差异无统计学意义，Western blot检测显示Cyclin D1蛋白表达在放疗后24 h明显升高。结论 下调VEGF表达可增加鼻咽癌细胞的放射敏感度，其机制可能是通过Cyclin D1信号通路途径使细胞周期发生G1/S期阻滞。Abstract: Objective To investigate the effect and possible mechanism of vascular endothelial growth factor (VEGF) down-regulated by small interfering RNA-mediated RNA interference (SiRNAi) on the radiosensitivity of nasopharyngeal carcinoma (NPC) cell line CNE-2. Methods pVEGF-siRNA (CNE-2 group), pNeg-siRNA (CNE-2/Neg-siRNA group), and CNE-2 cells transfected with VEGF and siRNA (CNE-2/VEGF-siRNA group) were constructed. Cell clonality was calculated by colony-forming unit assay after 6MVX-ray irradiation with 0, 2, 4, 6, 8, 10Gy respectively. The changes of cell cycle phase and apoptosis were determined using flow cytometry. Cyclin D1, Cyclin E, P16 and P53 were respectively measured by reverse transcription polymerase chain reaction (RT-PCR). Results Cell survival rate of CNE-2/VEGF-siRNA group was significantly decreased than those of CNE-2 and CNE-2/Neg-siRNA group after 6MV X-ray irradiation with 0, 2, 4, 6, 8, 10 Gy. All the values of radiobiological parameters including D0, Dq and SF2 in CNE-2/VEGF-siRNA group were respectively lower than those in CNE-2 and CNE-2/Neg-siRNA groups. Cell cycle in CNE-2/VEGF-siRNA group was arrested in the G1/S phase. Both VEGF mRNA and protein expression were significantly decreased in the experimental group compared with controls (P<0.05). The proliferation of treated CNE-2 cells was inhibited in vitro. Among Cyclin D1, Cyclin E, p16 and p53 gene, Cyclin D1 mRNA expression was increased significantly at 6, 12, and 24 h after irradiation. Western blot showed that Cyclin D1 protein expression was increased significantly at 24h after irradiation. Conclusion Down-regulating VEGF expression could enhance the radiosensitivity of NPC cells which mechanism might be arresting NPC cells in G1/S phase through Cyclin D1 signaling pathway.
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