卵巢癌中出核因子CRM1对p27^(kip1)亚细胞定位的影响

Effect of CRM1 on subcellular localization of p27^(kip1) in ovarian cancer

目的:研究出核因子CRM1参与p27kip1出核转运的机制,探讨其对p27kip1亚细胞定位的影响。方法:以人卵巢上皮细胞癌细胞株SKOV3及卵巢透明细胞癌细胞株ES2为研究对象,细胞周期同步化后用免疫荧光法测定不同细胞周期中CRM1、p27kip1和p-Ser10p27kip1蛋白的表达定位情况,并用Western Blot检查其在不同细胞生长周期的表达,最后运用免疫共沉淀法证实各分子之间的相互作用。结果:在G0/G1期卵巢癌细胞中,p27kip1定位表达于细胞核;在S期细胞中,p27kip1以pSer10p27kip1的形式存在于胞浆中,CRM1的表达水平与p-Ser10p27kip1相近。在G1期向S期转变的卵巢癌细胞中p27kip1与CRM1、p-Ser10-p27kip1的蛋白水平的表达趋势相反,p27kip1、p-Ser10p27kip1可分别与CRM1的结合来改变其在细胞中的定位。结论:出核因子CRM1通过与p27kip1结合的方式影响其核内外分布及表达水平,从而参与卵巢癌细胞周期调控。

Objective： To research the mechanism of CRM1 in transfer of p27kip1 outside of cell nucleus and its impact on the subcellular localization. Methods： Human ovarian cancer cell line SKOV3 and ovarian clear cell carcinoma cell line ES2 were selected as research objects. After cell cycle synchronization,immunofluorescence assay was used to detect the expressions and localizations of CRM1,p27kip1and p-Ser10p27kip1 in different cell cycles. Western Blot was used to detect their expressions during different cell cycles. Co-immunoprecipitation method was used to testify the interaction ofCRM1,p27kip1 and p-Ser10p27kip1. Results： p27kip1 protein localized in cell nucleus of ovarian cancer cells in G0 / G1 phase; p27kip1 protein localized in cytoplasm of ovarian cancer cells in S phase in the form of p-Ser10p27kip1; the expression level of CRM1 was similar to p-Ser10p27kip1. In ovarian cancer cells transforming from G1 phase to S phase,the expression tendency of p27kip1 was opposite to p-Ser10p27kip1 and CRM1, p27kip1 and p-Ser10p27kip1 could change their localizations in ovarian cancer cells by combining with CRM1. Conclusion： CRM1 can affect the distribution and expression of p27kip1 protein in and outside the cell nucleus by combining with p27kip1 in order to participate in cell cycle regulation of ovarian cancer.