Objective To investigate the mechanism of transcription regulation of GLI3 gene in idiopathic congenital talipes equinovarus.Methods pGL3-Gli3 luciferase report vectors were constructed,and the activity of Gli3 promoter was explored.A P-Match software was used to analyze the sequence upstream of the transcription start site of rat Gli3 gene,which was subsequently verified with chromatin immunoprecipitation assay (CHIP) and electrophoretic mobility shift assay (EMSA).Expression of the Gli3 gene was analyzed in L6 cells transfected with Hoxd13 small interference RNA(siRNA) and Hoxd13 expression vectors.Results The 5′ region of rat Gli3 gene contains two potential binding sites for the Hoxd13 protein.CHIP and EMSA assays both confirmed that Hoxd13 can directly bind with site 2.As shown in L6 cells,expression of Gli3 may be enhanced with silencing of Hoxd13,whilst exogenous expression of Hoxd13 can down-regulate transcription of Gli3.Conclusion Hoxd13 can directly regulate the expression of Gli3 gene through a Hoxd13 binding site in the limb of rat embryo.%目的 探讨在单纯性马蹄内翻足发生过程中GLI3基因的凋控机制.方法 构建荧光素酶报告基因表达载体,分析大鼠Gli3基因5′侧翼启动子区域的活性.用P-Match软件预测Gli3基因上游序列中转录因子的结合位点,并通过染色质免疫沉淀实验、凝胶迁移实验验证.用RNA干扰实验以及构建Hoxd13表达载体,观察其在L6细胞中对Gli3基因表达的影响.结果 在大鼠Gli3基因序列的启动子区域发现2个Hoxd13的结合位点,染色质免疫沉淀和凝胶迁移实验证实Hoxd13结合于结合位点2上.Hoxd13表达下调时,Gli3基因表达明显上调.Hoxd13基因表达上调时,Gli3基因则表达下调.结论 在大鼠胚胎肢体发育中,Hoxd13蛋白可能与Gli3基因启动子区的Hoxd13结合位点2结合,调控Gli3的表达.
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