Sp1介导雌激素上调LRP16基因表达的研究

Sp1 mediates estrogen-upregulated expression of LRP16 gene

目的:研究Sp1在雌激素上调LRP16基因表达中的作用。方法:采用ER,αSp1特异性单抗对雌激素作用不同时间点的MCF-7细胞进行染色质免疫共沉淀,snPCR扩增沉淀DNA上LRP16基因启动子区;化学合成Sp1小干扰RNA,与pS10荧光素酶报告重组子共转染MCF-7细胞,双荧光素酶测定法检测Sp1-S iRNA对LRP16基因表达的抑制作用。结果与结论:染色质免疫共沉淀结果提示Sp1蛋白直接结合于LRP基因-213 bp至-24 bp区域,雌激素激活的ERα增强其与DNA的结合力上调LRP16基因表达;应用Sp1小干扰RNA有效抑制了LRP16基因的雌激素反应性,明确了Sp1所结合的DNA顺式元件,从反面证实了Sp1蛋白对LRP16基因启动子区域递呈E2转激活活性的增强效应。

Objective： To explore the role of Spl in up-regulation of LRP16 gene expression by estrogen. Methods： Specific antibodies of ERa and Sp1 were used in chromatin immunoprecipitation assay of MCF-7 cells at different time points after estrogen （E2 ） treatment, and the promoter region of LRP16 gene was examined by semi-nested polymerase chain reaction（snPCR）. Hairpin small interference RNA（SiRNA） against Spl was synthesized by chemical method and transient-cotransfected with pS10 in MCF-7 cells. The knockdown endogenous LRP16 level was examined by luciferase assay. Results and Conclusion： Chromatin immunoprecipitation assay showed that Spl could bind to the LRP16 gene promoter region -213 bp to -24 bp, and hormonal regulation of LRP16 gene expression was linked to interactions of ERa and Spl with this promoter region. The Sp1-SiRNA down-regulated the LRP16 gene expression, suggesting that Spl protein enhances the E2-dependent activation effect of the human LRP16 gene promoter, and characterizes the element that associates Spl with the LRP16 gene.