体外沉默血管内皮生长因子对D-(-)-MTX/A549耐药细胞株迁移侵袭能力的影响

Effects of silencing VEGF gene expression on migration and invasion of D-(-)-MTX/A549 resistance cells

目的:研究血管内皮生长因子(vascular endothelial growth factor,VEGF)基因沉默对D-(-)-MTX/A549耐药细胞株迁移和侵袭的影响及可能的分子机制。方法:采用siRNA技术沉默D-(-)-MTX/A549耐药细胞株VEGF基因,细胞划痕试验和Transwell试验检测细胞迁移和侵袭能力,实时荧光定量PCR检测VEGF、MMP-2 mRNA,Western blot检测p-ERK1/2蛋白的表达。结果:VEGF siRNA序列及阴性对照成功转染至D-(-)-MTX/A549后,VEGF mRNA表达水平明显下降,与阴性对照相比下降了2.29倍,差异具有统计学意义(P=0.002),表明转染成功。阴性对照组与空白组差异无统计学意义(P>0.05)。转染后,D-(-)-MTX/A549迁移和侵袭能力明显下降(P值均<0.05),D-(-)-MTX/A549耐药细胞MMP-2 mRNA、p-ERK1/2的蛋白达也较阴性对照水平明显下降(P值均<0.05)。结论:抑制VEGF基因表达可以抑制D-(-)-MTX/A549细胞的迁移和侵袭能力,这一过程可能涉及到MMP-2和p-ERK1/2信号通路。

AIM： To study effects of silencing VEGF gene expression on migration and invasion of D-（-） -MTX/A549 resistance ceils. METHODS： VEGF siRNA was transfected to D-（-）-MTX/A549 resistance cells. Migration and invasion ablities were detected by scratch wound healing and transwell as- say respectively. The mRNA expression levels of VEGF and MMP-2 were examined by real-time fluo- rescent quantitative PCR. The protein expression level of p-ERK1/2 was detected by western blot a- nalysis. RESULTS：After VEGF siRNA and negative control were transfected to D- （-） - MTX/A549, VEGF mRNA expression level was decreased obvi- ously compared with negative control （ P = 0. 002 ）,negative control and blank groups have no statistical significance （P 〉 0.05 ）. D- （-） - MTX/A549 mi- gration and invasion ability significantly decreased （P 〈 0.05） ,MMP-2 mRNA and P-ERK1/2 protein levels were also significantly decreased after trans- fection （P 〈 0.05 ）. CONCLUSION ： Inhibition of VEGF expression can inhibit the D- （-） - MTX/ A549 cell migration and invasion ability, and this process may involve the MMP-2 and p-ERK1/2 sig- naling pathways.