RNA干扰HIF-2α基因对人肺腺癌A549细胞株侵袭转移的影响

Effect of RNA Interference Targeting HIF-2α Gene on Invasion and Proliferation of Lung Adenocarcinoma A549 Cell Line

目的:探讨小干扰RNA(small interfering RNA,siRNA)抑制缺氧诱导因子(hyposia-inducible factor 2α,HIF-2α)基因表达对人肺腺癌A549细胞株侵袭转移的影响。方法:构建针对HIF-2α-siRNA并转染A549细胞株,采用实时荧光定量多聚酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)和Western blot方法检测HIF-2α表达水平,通过Tran-swell试验及血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白检测,探讨HIF-2α-siRNA对A549细胞侵袭力的影响。结果:将HIF-2α-siRNA转染A549细胞后,HIF-2α的mRNA和蛋白表达水平均下调。qRT-PCR检测显示,第4号siRNA表达载体抑制作用最强,转染后24 h和48h对HIF-2α基因mRNA表达水平抑制率分别为(60.63±5.10)%和(80.00±3.55)%。Western blot检测同样证实,第4号siRNA表达载体对于HIF-2α蛋白表达抑制作用最强,转染后24 h和48 h抑制率分别为(31.69±11.56)%和(82.87±4.09)%。Transwell试验发现,转染HIF-2α-siRNA后,穿膜细胞数显著减少(P<0.05),表明细胞侵袭力下降。转染HIF-2α-siRNA后,VEGF表达水平显著下调(P<0.01)。结论:转染HIF-2α-siRNA可有效降低A549细胞的侵袭转移力。

Objective：To investigate the effect of small interfering RNA （siRNA） targeting hypoxi%inducible factor 2%（HIF- 2a） gene on the invasion and metastasis of A549 cell line. Methods：The siRNA eukaryotic expression vectors targeting HIF 2α gene were designed and transfected into A549 cell line. Quantitative real time PCR （qRT-PCR） and Western blot were used to detect mRNA and protein expression of HIF 2α gene. The migration of A549 cells was assayed using transwell cell culture chambers, and vascular endothelial growth factor（VEGF） protein expression was determined by enzyme-linked immunosorbent assay （EI.ISA）. Results： After transfection of HIF 2α-siRNA into A549, mRNA and protein expression of HIF-2αwere down regulated. The results of qRT-PCR showed that No. 4 siRNA vector was the most effective one in suppressing HIF-2α mRNA expression. Transfection of A549 with this siRNA vector resulted in sequence specific silencing with decreases of（60. 63 ± 5.10）% and （80.00 ± 3.55）% in HIF-2α mRNA transcription at 24h or 48h post-transfection. The Western blot test also demonstrated the best effectiveness of this expression vector with the inhibition rates of （31.69 ± 11.56） % at 24h post-trans- fection and （82.9 ± 4.09） % at 48h post-transfection. It was shown in migration analysis that the number of cells which had mi- grated through the filter was much amaller in groups treated with HIF-2α-siRNA than in control, which indicated a reduction in the invasive capacity of this group. ELISA analysis showed the protein expression of VEGF was significantly down-regulated when A549 cells were treated with siRNA targeting HIF-2αtransfection. Conclusions.. Eukaryotic expression vectors of siRNA which inhibit the expression of HIF-2α gene can effectively suppress the invasion and metastasis of A549 cells.