VEGFR-3 siRNA对人结肠癌细胞黏附力和侵袭性的抑制作用

Inhibitory effect of VEGFR-3-targeted siRNA against adherence and invasion of human colon cancer cells

目的:探讨VEGFR-3对人结肠癌细胞黏附力和侵袭性的影响。方法:构建携靶向VEGFR-3基因siRNA(small interfering RNA)表达载体,转染人结肠癌LoVo细胞,半定量RT-PCR和Western blotting检测转染前后LoVo细胞VEGFR-3 mRNA和蛋白表达的变化,基质-黏附实验检测细胞转染后的黏附能力,细胞侵袭实验检测转染后肿瘤细胞侵袭性的改变。结果:携靶向VEGFR-3基因siRNA的表达载体成功构建,RT-PCR检测转染siRNA后LoVo细胞VEGFR-3 mRNA表达水平降低;Western blotting检测转染siRNA后72 h LoVo细胞VEGFR-3蛋白表达下降,其表达相对值由(1.26±0.19)降至(0.39±0.12)(P<0.05)。转染siRNA 72 h后LoVo细胞的黏附能力显著下降[(0.626±0.047)vs(0.407±0.029),P<0.05];LoVo细胞穿膜细胞数(6.38±3.25)明显低于空白对照组(24.82±3.44)、非特异性对照组(23.58±3.73)(P<0.05)。结论:siRNA能够在LoVo细胞中引发RNA干扰效应,下调VEGFR-3基因的表达,进而抑制LoVo细胞的黏附能力和侵袭性。

Objective ： To construct a small interfering RNA （siRNA） expression vector （ psiRNA-VEGFR-3 ） targeting vascular endothelial growth factor receptor 3 （VEGFR-3） and to investigate the effects of VEGFR-3 siRNA on the adherence and invasion of human colon cancer cells. Methods： A siRNA expression vector （psiRNA-VEGFR-3） targeting VEGFR-3 were constructed and was used to transfect LoVo cells via lipofectamine 2000. The mRNA and protein expression of VEGFR-3 were examined after transfection by reverse transcriptase polymerase chain reaction （RT-PCR） and Western blotting, respectively. The tumor adhesion ability was detected by cell-matrix adhesion experiment and the invasion ability of tumor cells was evaluated by millicell chamber model. Results： The VEGFR-3 siRNA expression vector was successfully constructed. The expression of VEGFR-3 mRNA and protein was inhibited after psiRNA-VEGFR-3 transfection. Seven- ty-two hours after psiRNA-VEGFR-3 transfection, Western blotting assay showed that the expression of VEGFR-3 protein was decreased from （ 1.26 ±0.19） to （0.39 ±0.12） （P 〈0.05）, the adhesion ability of LoVo cells was also significantly decreased compared with the untransfected group and negative control group （0. 407 ± 0. 029 vs0. 626 ± 0. 047,0. 621 ± 0. 068, P 〈 0. 01 ）. The invasion assay demonstrated that the number of LoVo cells penetrating the membrane in the transfection group was significantly lower than those in the untransfected and negative control group （6.38 ± 3.25 vs 24.82 ± 3.44, 23.58 ± 3.73, P 〈 0.05 ）. Conclusion ： The siRNA of VEGFR-3 gene can effectively inhibit the mRNA and protein expression of VEGFR-3 in LoVo cells, therefore restraining the adhesion and invasion ability of LoVo cells.