GLI3、EN1基因在单纯性马蹄内翻足发生中的功能研究

为了探讨人类GLI3基因在单纯性马蹄内翻足(Idiopathic congenital talipes equinovarus, ICTEV)发生时所起的作用, 文章构建了大鼠Gli3基因启动子区域荧光素酶报告基因表达载体来分析Gli3基因启动子的活性, 并利用P-Match软件预测大鼠Gli3基因启动子区域可能的调控元件, 应用ChIP实验加以验证。并利用RT-PCR、免疫组化和Western blotting的方法分析大鼠En1与ICTEV的相关性。经P-Match软件预测, Gli3基因启动子区域有３个转录因子En1的可能结合位点, 经ChIP实验证实位点1是真正结合位点。RT-PCR、免疫组化和Western blotting方法都证实En1基因在马蹄内翻足模型鼠中表达下降。结果提示大鼠的转录因子 En1可能是Gli3基因的上游负调控元件。在ICTEV患者中, 很可能是由于EN1基因表达水平的下降导致了GLI3基因表达水平的上升, 最终导致ICTEV的发生。

The functions of GLI3 and EN1 genes in idiopathic congenital talipes equinovarus

To investigate the role of gene Gli3 in idiopathic congenital talipes equinovarus (ICTEV), we constructed the Gli3 luciferase reporter gene expression vectors to analyze the promoter activity of the rat gene Gli3. The regulatory element in the promoter region of the rat Gli3 was predicted using P-Match software and further verified by ChIP experiment. Meanwhile, the correlation between the rat En1 and ICTEV was evaluated by RT-PCR, immunohistochemistry, and Western blotting analyses. The result from P-Match software prediction showed that only one of the three possible En1 binding sites in Gli3 promoter region was interacted directly with En1 in vivo, which was confirmed by ChIP analysis. The results from RT-PCR, immunohistochemistry and Western blotting analyses suggested that En1 was down-regulated in ICTEV model rats compared to the controls. Our results indicated that En1 might be the negative regulatory element in the upstream of Gli3. The low expression level of EN1 in ICTEV could contribute to the up-regulation of GLI3, which led to the genesis of ICTEV.