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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: To observe the transcription of Gli1, a transcription factor of estrogen receptor ERα, in human breast cancer cell line MCF-7 based on the previous study that the downstream transcription factor Gli1 of Hedgehog signaling pathway can inhibit the estrogen signaling pathway in breast cancer cells Activity and expression, to explore whether there is interaction between the two signaling pathways. Methods: Transfection of Gli1 expression plasmid and Gli reporter plasmids pGli-BS-luc and ERα expression plasmid or empty vector into MCF-7 breast cancer cells by transient transcriptional activity assay , Observe the change of Gli1 transcriptional activity. Then, the effect of ERα overexpression on Gli1 mRNA and protein expression in breast cancer cells was detected by real-time quantitative PCR and Western blot respectively. Results: The luciferase activity increased with the increase of overexpression of ERα. The luciferase activity increased to 3.5 times that of control cells (P <0.001) when ERα was 500 ng / well. After estrogen treatment, there was no significant change in luciferase activity. Overexpression of ERα increased the expression of Gli1 mRNA by 2 folds (P <0.01), and also significantly increased the expression of Gli1 protein. Conclusion: ERα overexpression in MCF-7 cells can significantly enhance the transcription activity of Gli1 and increase the expression of Gli1. This indicates that there is an interaction between the transcription factor ERα and Gli1 in breast cancer cells.