HIF-1α介导低氧诱导的内皮细胞Brm表达上调的机制研究

摘要

目的: 本研究拟探讨低氧诱导因子1α(HIF-1α)在低氧诱导的血管内皮细胞Brm表达上调中的作用及机制,为低氧性肺动脉高压的防治提供理论依据.方法: 在内皮细胞中过表达或siRNA干扰HIF-1α的表达,并给予1%低氧处理24 h后,运用萤光素酶报告基因检测方法检测Brm启动子的转录活性,运用RT-qPCR检测Brm的mRNA表达水平,运用Western blot检测Brm的蛋白表达水平.运用ChIP技术检测低氧条件下HIF-1α与Brm启动子区域的结合变化情况.结果: 过表达HIF-1α可显著上调Brm的启动子活性、mRNA及蛋白表达,而siRNA干扰HIF-1α可显著抑制Brm的启动子活性、mRNA及蛋白表达,ChIP实验揭示低氧可显著增强HIF-1α与Brm启动子的结合.结论: 低氧上调内皮细胞中Brm的表达依赖HIF-1α的转录调控作用.%AIM: To clarify the molecular mechanism of Brm activation in endothelial cells during hypoxia and hypoxic pulmonary hypertension.METHODS: Hypoxia-inducible factor 1 alpha (HIF-1α) was over-expressed or depleted in the endothelial cells under hypoxia.Luciferase activity was assayed after transfection using a luciferase reporter assay system.The expression of Brm at mRNA and protein levels was detected by RT-qPCR and Western blot.The occupancy of HIF-1α on Brm promoter was detected by the method of chromatin immunoprecipitation (ChIP).RESULTS: Brm expression was induced in cultured endothelial cells challenged with hypoxia.Over-expression of HIF-1α enhanced, while the depletion of HIF-1α attenuated Brm transactivation and expression under hypoxia.The results of ChIP revealed that hypoxia up-regulated the occupancy of HIF-1α on Brm promoter.CONCLUSION: HIF-1α, which is induced by hypoxia, binds to Brm promoter, and further promotes Brm transactivation in endothelial cells.