雌激素受体α在乳腺癌细胞中对Gli1转录活性和表达的影响

Effect of estrogen receptor α on transactivation and expression of Gli1 in breast cancer cells

目的:在先前研究发现Hedgehog信号通路的下游转录因子Gli1能够抑制乳腺癌细胞中的雌激素信号通路活性的基础上,观察雌激素受体ERα在乳腺癌细胞MCF-7中对转录因子Gli1的转录活性和表达的影响,以探讨两条信号通路之间是否存在着交互作用。方法:应用荧光素酶报告基因转录活性分析的方法,将Gli1的表达质粒和Gli的报告基因质粒pGli-BS-luc以及ERα的表达质粒或者空载体共同瞬时转染到乳腺癌细胞MCF-7中,观察Gli1转录活性的变化。然后分别用实时定量PCR和蛋白质印迹的方法检测乳腺癌细胞中ERα的过表达对Gli1 mRNA和蛋白表达的影响。结果:荧光素酶的活性随着ERα过表达剂量的增加而增加,ERα的质粒量在500 ng/孔时可将荧光素酶的活性升高到对照细胞的3.5倍(P<0.001)。雌激素处理后,荧光素酶的活性无显著变化。过表达ERα后可将Gli1 mRNA的表达水平提高为原来的2倍(P<0.01),也可明显增加Gli1蛋白的表达水平。结论:ERα在MCF-7细胞中的过表达能够明显增强Gli1的转录活性,增加Gli1的表达。这表明乳腺癌细胞中,转录因子ERα和Gli1之间存在着交互作用。

Objective：It has been found that the expression of Hedgehog signaling molecule Gli1 can inhibit the activity of estrogen signaling pathway.The present study is to observe the effect of estrogen receptor α（ERα） on the transactivation and expression of Gli1 in breast cancer cells,so as to study whether there is a cross talk between the two pathways.Methods： Using luciferase reporter gene transactivation analysis,we cotransfected MCF-7 cells with pGli-BS-luc,pcDNA3.1-Gli1,pRL-CMV,pSG5-ERα or equimolar amounts of pSG5 vector.Then the cells were subjected to Luciferase Assays to analyze the change of Gli1 transactivation.The mRNA and protein expression of Gli1 following ectopic overexpression of ERα was also analyzed by quantitative real-time PCR and Western blotting analysis.Results： Expression of ERα induced the luciferase activity in a dose-dependent manner.ERα at 500 ng/well increased the activity of luciferase by 3.5 folds（P〈0.001）.The luciferase activity had no obvious changes after estrogen treatment.Overexpression of ERα increased the expression of Gli1 mRNA by 2 folds（P〈0.01）,and obviously increased the expression of Gli1 protein.Conclusion： Overexpression of ERα in MCF-7 cells can greatly increase the transactivity of Gli1 and increase its expression,which indicates that there is a cross talk between the two transcription factors in breast cancer cells.