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目的明确介导AngⅡ调控人内皮细胞中巨噬细胞移动抑制因子(migration inhibitory factor,MIF)表达的转录因子。方法构建长度依次递减的MIF基因5’调控区的荧光报告基因表达载体,并将其转化入人脐静脉内皮细胞EAhy926。利用双荧光基因报告系统检测AngⅡ诱导后MIF基因5’调控区不同区段的转录活性。通过突变转录活性高的MIF5’调控区序列,初步确定可能的转录因子结合序列。设计针对可能的转录因子的小干扰RNA(siRNA)序列,并构建其表达载体,分别检测候选转录因子沉默的HEAY926中MIF的转录活性和表达水平。结果双荧光基因报告检测显示,AngⅡ调控MIF表达的高转录活性区段位于-507至-188之间。序列突变和荧光素酶活性分析显示,MIF基因5’调控区-350至-339间的AP-1结合序列决定了MIF基因的转录活性。AP-1沉默后的HEAY926中AngⅡ调控的MIF转录活性显著降低(P<0.05),MIFmRNA和蛋白的表达水平也显著降低(P<0.05)。结论AngⅡ通过转录因子AP-1调控内皮细胞中MIF的表达。
Objective To express the transcription factor that mediates the regulation of macrophage migration inhibitory factor (MIF) expression in human endothelial cells. Methods The fluorescent reporter gene expression vector of 5 ’regulatory region of MIF gene with decreasing length was constructed and transformed into human umbilical vein endothelial cells EAhy926. Double-fluorescent gene reporter system was used to detect the transcriptional activity of different regions of the 5 ’regulatory region of MIF gene induced by AngⅡ. By mutating MIF5 ’regulatory region sequences with high transcriptional activity, preliminary possible transcription factor binding sequences were identified. Small interfering RNA (siRNA) sequences targeting potential transcription factors were designed and their expression vectors were constructed to detect the transcriptional activity and expression levels of MIF in HEAY926 cells silenced by candidate transcription factors. Results Double-fluorescent gene reporter assay showed that AngⅡregulated the transcriptional activity of MIF in a region between -507 and -188. Sequence analysis of mutations and luciferase activity revealed that the AP-1 binding sequence between the -350 to -339 region of the 5 ’regulatory region of MIF gene determines the transcriptional activity of the MIF gene. AngⅡ-regulated MIF transcriptional activity was significantly decreased (P <0.05) and MIF mRNA and protein expression was significantly lower in AP-1-silenced HEAY926 cells (P <0.05). Conclusion AngⅡ regulates the expression of MIF in endothelial cells through the transcription factor AP-1.