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[目的]探讨缺氧诱导因子-1α(HIF-1α)对Snail启动子活性的作用,并鉴定其作用位点。[方法]应用transfac软件分析Snail启动子区域潜在的HIF-1α结合位点。通过荧光素酶报告基因载体、电泳迁移率实验(EMSA)和染色质免疫沉淀(Ch IP)实验研究HIF-1α是否能够与Snail启动子区域潜在的位点结合,直接调控Snail的表达。[结果]生物信息学分析发现Snail启动子区域存在2个HIF-1α可能的结合位点(-653,-649)和(-543,-538)。双荧光素酶报告基因检测发现(-543,-538)位点是HIF-1α特异性结合于Snail的位点,HIF-1α蛋白与之结合后调节Snail基因转录活性。EMSA和Ch IP实验证实了HIF-1α蛋白和Snail启动子区(-543,-538)结合,直接上调Snail的表达。[结论]Snail启动子区域存在HIF-1α的结合位点,HIF-1α与之结合后直接调控Snail的表达。
[Objective] To investigate the effect of hypoxia-inducible factor-1α (HIF-1α) on Snail promoter activity and to identify its site of action. [Method] The potential HIF-1α binding site in Snail promoter region was analyzed by transfac software. The luciferase reporter gene vector, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments were used to investigate whether HIF-1α could bind to potential sites in the Snail promoter region and directly regulate Snail expression. [Results] Bioinformatics analysis revealed two possible binding sites (-653, -649) and (-543, -538) of HIF-1α in Snail promoter region. Dual luciferase reporter assay revealed that the (-543, -538) site of HIF-1α specifically binds to Snail, and the HIF-1α protein binds to it and regulates the transcriptional activity of Snail. EMSA and ChIP experiments confirmed that the HIF-1α protein binds to the Snail promoter region (-543, -538) and directly up-regulates Snail expression. [Conclusion] There is HIF-1α binding site in the Snail promoter region. HIF-1α directly controls the expression of Snail.