雌激素在乳腺癌细胞中通过雌激素受体-ɑ调节PES1的表达及其稳定性的体外实验研究

Estrogen regulates PES1 expression and its stability through estrogen receptor-ɑof the breast cancer cells in vitro

目的:阐明PES1在乳腺癌细胞株中的表达情况以及PES1与雌激素受体-ɑ(estrogen receptor-ɑ,ER-ɑ)的关系。方法:培养不同的乳腺癌细胞系BICR、ZR75,采用免疫荧光法检测PES1的表达及定位,并通过顺势转染RNA干扰技术抑制PES1的表达,利用平板克隆形成实验和MTT实验检测细胞的生长增殖表型改变。同时,用不同浓度梯度及不同时间点的雌激素刺激相关乳腺癌细胞,用蛋白印迹法检测PES1的蛋白表达水平。最后,利用雌激素刺激上述细胞并通过RNA干扰抑制ER-ɑ后,使用蛋白印迹法检测PES1蛋白表达水平,同时,在放线菌酮的作用下,进一步检测乳腺癌细胞中PES1与ER-ɑ之间的相关性和稳定性,并进一步检测ER-ɑ对PES1的泛素化水平的影响。结果:我们通过免疫荧光发现PES1在几种乳腺癌细胞中均表达,并进一步证明干扰PES1可以抑制乳腺癌细胞克隆的形成及生长。此外,我们在乳腺癌细胞中发现雌激素主要通过其受体ER-ɑ提高PES1的蛋白水平,进一步发现雌激素对PES1转录的影响并不明显,而可能是通过ER-ɑ影响PES1的泛素化水平进而增强PES1蛋白的稳定性。结论:对于乳腺癌细胞,内源性ER-ɑ和PES1在乳腺癌的发生发展中相互协调发挥着重要作用。

Objective:To clarify the PES1 expression in breast cancer cell lines as well as the relationship between PES1 and estrogen receptor-ɑ(ER-ɑ).Methods:The breast cancer cell lines BICR and ZR75 were cultured.PES1 expression and localization were detected using immunofluorescence.PES1 expression was inhibited by homeopathic transfection RNA interference technology.The growth and proliferation phenotypic changes of cells were detected by plate cloning assay and MTT assay.At the same time,estrogen at different concentration gradients and different time points was used to stimulate related breast cancer cells,and the expression level of PES1 was detected by Western blot.Finally,estrogen was used to stimulate the above cells and ER-ɑwas inhibited through RNA interference.The expression level of PES1 protein was detected by Western blot.At the same time,under the action of actinomycin,the correlation and stability between PES1 and ER-ɑin breast cancer cells were detected,and the effect of ER-ɑon the ubiquitination level of PES1 was detected.Results:We found that PES1 was expressed in several breast cancer cells through immunofluorescence,and further proved that interference with PES1 can inhibit the clone formation and growth of breast cancer cells.In addition,we found that estrogen can improve the protein level of PES1 through ER-ɑin breast cancer cells.It was further found that estrogen had no significant effect on PES1 transcription,but probably affected the ubiquitination level of PES1 through ER-ɑ,thereby enhancing the stability of PES1 protein.Conclusion:For breast cancer cells,endogenous ER-ɑand PES1 play an important role in the occurrence and development of breast cancer.