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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 study the mechanism of nuclear factor CRM1 involved in the nuclear export of p27kip1 and to explore its effect on the localization of p27kip1 subcellular localization. Methods: Human ovarian epithelial cell line SKOV3 and ovarian clear cell carcinoma cell line ES2 were used as research objects. The expression and localization of CRM1, p27kip1 and p-Ser10p27kip1 in different cell cycles were detected by immunofluorescence after cell cycle synchronization. Western Blot was used to examine its expression in different cell cycle. Finally, co-immunoprecipitation was used to confirm the interaction between the molecules. Results: In G0 / G1 ovarian cancer cells, p27kip1 was localized in the nucleus. In S phase cells, p27kip1 was present in the cytoplasm in the form of pSer10p27kip1, and the expression level of CRM1 was similar to that of p-Ser10p27kip1. The expression of p27kip1 and CRM1 and p-Ser10-p27kip1 protein in ovarian cancer cells with G1 phase to S phase were opposite. The expression of p27kip1 and p-Ser10p27kip1 could be changed respectively with CRM1 to change its localization in cells. Conclusion: The nuclear factor CRM1 is involved in the cycle regulation of ovarian cancer cells by affecting the distribution and expression level of nuclear factor CRM1 through binding to p27kip1.