VEGF-C siRNA载体构建及其对转染食管癌细胞中VEGF-C表达的影响

Construction of VEGF-C siRNA expression vector and its inhibitory effects on the expression of VEGF-C in EC9706 cells

目的观察小分子干扰RNA(siRNA)对人食管癌细胞EC9706中血管内皮生长因子C(VEGF-C)表达的抑制作用。方法根据siRNA设计原则,针对人VEGF-C cDNA序列设计并合成编码siRNA的寡核苷酸序列,并将其克隆入siRNA表达载体,构建重组质粒。将重组质粒转染入EC9706细胞株,通过免疫组织化学、RT-PCR和原位杂交法检测转染细胞中VEGF-C蛋白和mRNA的表达。结果正常未转染(对照组)和无关序列转染(无关序列组)EC9706中,均有大量VEGF-C蛋白阳性棕色颗粒、mRNA阳性条带和紫蓝色阳性颗粒,而在siRNA转染EC9706(siRNA_(1～3)组)中,仅有少量弱表达颗粒和较弱的阳性条带,与对照组和无关序列组相比,P均<0.01。结论成功构建了VEGF-C siRNA载体。此载体能有效抑制人食管癌细胞EC9706中VEGF-C的表达,其可能成为抗食管癌淋巴管生成的一种新方法。

Objective To examine the inhibition of vascular endothelial growth factor C （VEGF-C） by small interfering RNA in human esophageal cancer EC9706 cells. Methods According to the encoding sequences of mRNA of VEGF- C, three pairs of oligonucleotide sequence were designed, synthesized and cloned into siRNA expression vector. The recombinant VEGF-C-targeted siRNA eukaryotic expression plasmids were transfected into esophageal carcinoma EC9706 ceils via lipofectin mediation. Expressions of VEGF-C protein and mRNA in EC9706 cells were assayed by SP immunohistochemistry, semi-quantitative RT-PCR and in situ hybridization before or after the stable transfection, respectively. Results There were lots of positive granules and positive bands in normal EC9706 cells and in the transfection group cells with transfected not-matching any known human coding mRNA. The transfection group ceils with transfected by VEGF-C-targeted siRNA expressed few positive granules and weak positive bands. There was significant difference between them （ P 〈 0.01 ）. Conclusions siRNA of VEGF-C was constructcd. The specific siRNA of VEGF-C can significantly inhibit the expression of VEGF-C in EC9706 ceils. The siRNA against VEGF-C maybe serve as a potential method for cancer treatment in human esoohazeal cancer.