靶向血管内皮生长因子受体3基因的小干扰RNA表达载体构建及其生物学效应

Construction of small interfering RNA expression vector targeting VEGFR-3 gene and its biologic effects

目的构建靶向血管内皮生长因子受体3(VEGFR-3)基因的小干扰RNA(siRNA)表达载体(psiRNA-VEGFR-3),并观察其抑制人结肠癌细胞株LoVo细胞中VEGFR-3基因表达的效果及生物学效应。方法设计并合成1对VEGFR-3编码基因的反向重复序列,定向克隆至载体pSUPER,构建siRNA真核表达载体,利用脂质体2000将构建的psiRNA-VEGFR-3载体转入LoVo细胞中,应用半定量RT-PCR检测基因转染前后VEGFR-3mRNA表达水平的变化,四甲基偶氮唑蓝法观察细胞生长变化、凋亡率及细胞周期的变化。结果成功构建针对VEGFR-3基因的siRNA表达载体。psiRNA-VEGFR-3可显著抑制LoVo细胞中VEGFR-3基因的表达(P<0.01);转染psiRNA-VEGFR-3后能够诱导LoVo细胞发生明显凋亡。结论psiRNA-VEGFR-3载体能够在LoVo细胞中引发RNA干扰效应,通过下调VEGFR-3基因表达可以抑制细胞增殖,诱导LoVo细胞发生明显凋亡。

Objective To construct the small interfering RNA （siRNA） expression vector （psiRN- AVEGFR-3） targeting vascular endothelial growth factor receptor 3 （VEGFR-3） gene and to observe its suppressive effect on the expression of VEGFR-3 gene in human colon cancer cell line LoVo and its biologic effects. Methods A pair of reverse repeat of VEGFR-3 gene encoding was designed and synthesized, colonized to pSUPER vector, and siRNA eukaryotic expression vector was constructed. The psiRNA-VEGFR-3 vector was transfected into LoVo ceils by Lipofectamine 2000, and the mRNA expression of VEGFR-3 was detected by semi-quantitative reverse transcription-polymerase chain reaction before and after transfection. Cell growth was measured by methyl thiazolyl tetrazolium colorimetry, and apoptotic ratio and cell cycle were detected by flow cytometry. Results psiRNA-VEGFR-3 vector was successfully constructed and it could significantly inhibit the expression of VEGFR-3 gene in LoVo cells （P 〈0.01）. Transfection of psiRNA-VEGFR-3 vector could also induce significant apoptosis of LoVo cells. Conclusion psiRNA-VEGFR-3 vector can trigger RNA interference effect in LoVo cells, and it can inhibit cell proliferation and induce apoptosis of LoVo cells by downregulation of VEGFR-3 gene expression.