摘要目的 以特异性siRNA在转录水平下调VEFG表达,观察对人鼻咽癌细胞株(CNE2)生长、转移等生物学活性的影响并探讨其可能分子机制.方法 构建和筛选VEGF-siRNA高效干扰质粒并测序鉴定,转染研究细胞株,观察干预VEGF表达对鼻咽癌细胞增殖、细胞周期及转移的影响.结果 以siRNA转染CNE2细胞,VEGF在基因转录和蛋白水平表达明显降低,癌细胞增殖明显受抑并呈时间依赖性;干预组癌细胞平板克隆形成能力明显降低、细胞失巢凋亡率显著增加,癌细胞增殖周期发生G1期阻滞,CyclinD1及p-Erk表达降低.结论 干预VEGF基因转录可经Erk/MAPK信号通路抑制鼻咽癌细胞增殖等生物学行为,提示VEGF为潜在的鼻咽癌基因治疗的有效靶目标.

abstractsObjective To investigate down-regulation of vascular endothelial growth factor (VEGF) gene at mRNA level on effects of nasopharyngeal carcinoma (NPC) cell proliferation and analyze the key molecule expressions of extracellular-signal regulated protein kinase/mitogen-activated protein kinase (Erk/MAPK) pathway to explore its molecular mechanism.Methods Highest-efficiency VEGF-siRNA plasmid was successfully constructed and screened,confirmed by sequencing,and then transfected to CNE2 cell line.Interference efficiency was quantitatively evaluated by a fluorescence quantitation polymerase chain reaction assay.Expressions of VEGF,cell cycle regulator cyclin D1,and key molecules in Erk/MAPK pathway were analyzed with Western blot.Cell proliferation was tested by cell counting kit-8 (CCK-8) kit.Clone formation rate was checked with plate clone formation assay.Alteration of cell cycle was measured with flow cytometry.Stable cell clones were screened by G418.Results VEGF expression in NPC cells transfected with specific siRNA were significantly down-regulated at the levels of mRNA or protein,and cell proliferations were inhibited in a time-dependent manner,with significant decreases of colony-forming,cyclinD1,and p-Erk,and with cell cycle arrested at G1 phase.Conclusions Intervencing VEGF gene transcription inhibited NPC cell proliferation through Erk/MAPK signaling pathway,and suggesting that VEGF should be a novel therapeutic target for NPC.