RNA干扰VEGFR-2表达对Calu-1细胞增殖、迁移、侵袭力及放射效应影响

目的 研究VEGFR-2对肺癌细胞系Calu-1细胞增殖、迁移、侵袭及联合放射后凋亡率影响, 并探讨其可能机制。方法 siRNA敲低Calu-1细胞中的VEGFR-2基因, 借助实时荧光定量PCR和蛋白印迹法检测VEGFR-2表达水平的变化;将细胞分为对照组、VEGF组、VEGFR-2基因敲低组和基因敲低加VEGF组。利用CCK8法、细胞划痕实验及Transwell实验分别检测细胞增殖、迁移和侵袭能力变化, 利用蛋白印迹法检测VEGFR-2及下游相关信号通路蛋白表达水平变化;各组细胞联合放射后, 检测细胞凋亡。结果 RNA干扰VEGFR-2后Calu-1细胞中VEGFR-2的mRNA水平和蛋白水平均降低(P=0.001、0.000);RNA干扰VEGFR-2后Calu-1细胞的增殖、迁移、侵袭能力均降低(P=0.000、0.000、0.031);RNA干扰VEGFR-2后Calu-1细胞中Akt、ERK1/2、p38的蛋白磷酸化水平均降低(P=0.336、0.986、0.553);RNA干扰VEGFR-2后联合放射后Calu-1细胞的凋亡率增加(P=0.012), RNA干扰VEGFR-2后HIF-1α蛋白表达被抑制(P=0.016)。结论 VEGFR-2基因表达敲低后显著抑制了Calu-1细胞的多项生理功能, 并提高了放射后细胞凋亡率。

Effects of RNA interference-mediated silencing of vascular endothelial growth factor receptor-2 on proliferation,migration, invasion,and radiation-induced effects in Calu-1 cells

Objective To investigate the effects of vascular endothelial growth factor receptor-2(VEGFR-2) on proliferation, migration, invasion, and apoptosis after radiotherapy in lung cancer cell line Calu-1, and to explore the probable mechanisms. Methods Small interference RNA (siRNA)-mediated silencing of VEGFR-2 gene was performed on Calu-1 cells, and the mRNA and protein expression of VEGFR-2 was determined by quantitative real-time PCR and Western blot, respectively. The cells were divided into control group, vascular endothelial growth factor (VEGF) group, VEGFR-2 specific siRNA (siKDR) group, and siKDR+VEGF group. The changes in proliferation, migration, and invasion were evaluated by the CCK8 assay, cell scratch wound-healing assay, and transwell migration assay, respectively. The protein expression of VEGFR-2 and proteins in the related downstream signaling pathway was measured by Western blot. Apoptosis in each group was determined after radiotherapy. Results After RNA interference-mediated silencing of VEGFR-2, the mRNA and protein expression of VEGFR-2 was significantly reduced (P=0.001, P=0.000);the proliferation, migration, and invasion of Calu-1 cells were also significantly reduced (P=0.000, P=0.000, P=0.000);the phosphorylation levels of AKT, ERK1/2, and p38 were significantly reduced in Calu-1 cells (P=0.336, P=0.986, P=0.553);the apoptosis in Calu-1 cells was significantly elevated (P=0.0012);the protein expression of HIF-1α was significantly inhibited (P=0.016). Conclusions The VEGFR-2 gene silencing significantly inhibits several physiological functions of Calu-1 cells and elevates the apoptosis rate after radiotherapy.