雌激素受体α上调前列腺癌细胞株LNCaP中L-Plastin启动子转录活性研究

Estrogen receptor alpha up-regulates transcription activity of L-plastin promoter in prostate cancer cell line LNCaP

目的鉴定雌激素受体在L-Plastin启动子上的结合位点,进一步阐明雌激素受体在激素依赖型前列腺癌中的作用机制。方法利用TF SEARCH软件分析L-Plastin启动子的序列,寻找可能调控L-Plastin表达的雌激素受体结合位点,利用PCR定点突变法构建删除该转录因子结合位点的重组子,检测切除该片段序列后荧光素酶活性,并用凝胶滞后实验和超滞后实验证实雌激素受体是否结合于该位点,研究雌激素受体在激素依赖型前列腺癌中调控L-Plastin表达的作用。结果TF SEARCH软件分析表明,在L-Plastin启动子上距转录起始点-85^-70 bp处,含有雌激素受体结合位点ATTTCACTGTGACCT,删除该结合位点后L-Plastin启动子荧光素酶活性明显下降,凝胶滞后实验和超滞后实验表明雌激素受体α是结合于该位点的转录因子。结论雌激素受体α具有促进L-plastin在激素依赖型前列腺癌中表达的作用。

Objective To identify the binding site of estrogen receptor on L-Plastin promoter in prostate cancer cell line LNCaP and partly elucidate the mechanism of estrogen receptor regulating prostate cancer. Methods TF SEARCH software was used to analyze the possible binding site of estrogen receptor in L-Plastin promoter, PCR Site-Mutagenesis technique was performed to delete the binding site of estrogen receptor and Luciferase activity assay was carried out after deletion of the binding site. Gel Shift Assay and Supershift Assay were used to confirm estrogen receptor binding the speculated estrogen receptor elements. Results Estrogen receptor binding site ATTTCACTGTGACCT, which located at 85-70 bp of L-Plastin promoter, was identified with the TF SEARCH software. After deletion of the estrogen receptor binding-site, luciferase activity of L-Plastin promoter was obviously reduced. Gel shift assay and supershift assay revealed that estrogen receptor α was the transcription factor binding ATTTCACTGTGACCT. Conclusion Estrogen receptor α plays an important role in the up-regulation of L-Plastin expression in hormone-dependent prostate cancer cell line LNCaP.