高级搜索

siRNA 沉默HIF-1α在缺氧状态下对食管鳞癌细胞VEGF表达的影响

Effcet of Silencing HIF-1α by siRNA on Expression of Vascular Endothel ial Growth Factor in Esophageal Squamous Cell Carcinoma Cell under Hypoxia

  • 摘要: 目的 观察体外乏氧培养条件下食管鳞癌细胞系EC9706中HIF-1α和VEGF的表达,探讨HIF-1α在低氧条件下对食管鳞癌血管生成的调控作用。方法 CoCl2化学缺氧法模拟肿瘤缺氧环境,RT-PCR、免疫组化法和免疫印迹法分别检测缺氧状态下HIF-1α和VEGF在mRNA和蛋白水平的表达。采用化学合成小干扰RNA(siRNA)介导的RNA干扰技术(RNAi)用siRNA转染EC9706细胞。观察转染后HIF-1α沉默效果。结果 低氧条件下,EC9706细胞HIF-1amRNA水平稳定,蛋白表达显著升高,而VEGFmRNA和蛋白的表达均显著升高。SiRNA转染EC9706后能够显著下调HIF-1α的基因表达,同时VEGF基因的表达也受到明显抑制。结论 缺氧促使EC9706细胞HIF-1α在蛋白水平表达升高,并通过转录激活VEGF的机制调控食管鳞癌血管生成。

     

    Abstract: Objective  To investigate the expression of HIF-1 and vascular endothelial growth factor (VEGF) in human esophageal squamous cell carcinoma cell line EC9706 under hypoxia. To observe the effect of HIF-1αon hypoxia2activatad angiogenesis regulation pathway in esophageal squamous cellcarcinoma. Methods  CoCl2 was used as a chemical hypoxiainducible reagent to mimic tumor hypoxic microenviroment . mRNA and protein levels of HIF-1α and VEGF were detected by semiquantitative reverse transceiption-polymerase chain reaction ( RT-PCR) and immunohistochemistry. With RNA interference (RNAi) originated by small interference RNA ( siRNA ) to use siRNA transfected EC9706 cells. Western-blot was used to detect gene scilencing effect on HIF-1α. RT-PCR and immunohistochemistry were used to observe the change of VEGF gene expression af ter HIF-1αgene silence. Results  Under hypoxia, mRNA level of HIF-1α was stable, while its protein level increased obviously. Both mRNA and protein levels of VEGF were up-regulated. The siRNA targeting HIF-1α gene down-regulated HIF-1 gene in EC9706 cells efficiently, and VEGF gene was down-regulated as well. Conclusion  Hypoxia can increase protein level of HIF-1αin esophagus. HIF-1α up-regulates the gene expression of VEGF which promotes angiogenesis in esophagus under hypoxic microenviroment .

     

/

返回文章
返回