siRNA沉默HIF-2α对缺氧培养的BGC-823胃癌细胞中VEGF表达的影响

Effect of Silencing HIF-2α by siRNA on Expression of Vascular Endothelial Growth Factor in BGC-823 Gastric Adenocarcinoma Cell under Hypoxia

目的:观察乏氧培养条件下胃腺癌细胞系BGC-823中HIF-2α和VEGF的表达,探讨HIF-2α在低氧条件下对胃癌血管生成的调控作用。方法:采用实时定量聚合酶链反应(Real-time PCR)和Western blot法分别检测低氧状态下HIF-2α和VEGF在mRNA和蛋白水平的表达。进一步采用化学合成小干扰RNA(siRNA)介导的RNA干扰技术(RNA interference,RNAi)转染BGC-823细胞。观察转染后HIF-2α沉默效果。结果:与常氧时比较,缺氧后BGC-823细胞HIF-2α、VEGF的mRNA水平稳定,而蛋白的表达水平均明显升高(P<0.05),siRNA转染BGC-823后能够显著下调HIF-2α的基因表达,同时VEGF基因的表达也受到明显抑制。结论:缺氧可促使BGC-823细胞HIF-2α在蛋白水平表达升高,并通过激活VEGF的机制调控胃癌血管生成,切断HIF-2α途径可能会有效阻止胃癌血管生成。

Objective： To investigate the effect of specific silencing hypoxia inducible factor-2 alpha （HIF-2α） by small interference RNA（siRNA） on the expression of vascular endothelial growth factor in BGC-823 gastric cancer cells under hypoxia. To observe the effect of HIF-2α on hypoxia-activated angiogenesis regulation pathway in gastric carcinoma. Methods： BGC-823 gastric adenocarcinoma cell line acts as the research subject by purchasing. We used chemical hypoxia inducer cobalt chloride （ CoC12 ） as a chemical hypoxia-inducible reagent to stimulate tumor hypoxic micro-environment. mRNA and protein levels of HIF-2α and VEGF were detected by Quantitative Real-time PCR and immunohistochemistry. RNA interference （RNAi） technology was performed to transfect BGC-823 cells with HIF-2α siRNA. Results： Under hypoxia, protein levels of HIF-2α and VEGF were increased obviously. The siRNA targeting HIF-2α gene down-regulated HIF-2α gene in cells efficiently, and VEGF gene was down-regulated as well. Conclusion： Hypoxia can increase the expression level of HIF-2α gene in BGC-823 cell. HIF-2α can activate the downstream target gene--VEGF which promotes angiogenesis in gastric cancer under hypoxic microenvironment.