Ubiquitin B在HSP90抑制剂17-AAG诱导人宫颈癌HeLa细胞周期阻滞中的作用

Role of Ubiquitin B in 17-AAG-Induced Cell Cycle Arrest in HeLa Cells

目的 研究Ubiquitin B（Ubb）在热休克蛋白90（HSP90）抑制剂17-AAG诱导人宫颈癌HeLa细胞周期阻滞中的作用及机制.方法 不同浓度17-AAG处理HeLa细胞后,流式细胞术检测细胞周期分布,荧光分光光度法检测细胞蛋白酶体活性变化;Ubb siRNA 转染HeLa细胞后,Real Time PCR法检测Ubb干扰效应,Western 印迹检测细胞周期相关蛋白的表达改变.结果 17-AAG可以诱导HeLa细胞阻滞于G2/M期,同时显著增强细胞内糜蛋白酶样蛋白酶体活性,并且两者的变化均呈现剂量依赖性;干扰HeLa细胞内Ubb后,可以逆转17-AAG引起的G2/M期阻滞;17-AAG可明显下调HeLa细胞周期相关蛋白Cdk1和Hec1的表达,并且这一变化也是Ubb依赖的.结论 Ubb在17-AAG诱导的HeLa细胞周期阻滞中发挥重要作用,Ubb和HSP90抑制剂17-AAG在功能上相互关联,可能成为宫颈癌治疗的新靶点.

Objective To explore the possible mechanism of Ubiquitin B in 17-AAG-induced cell cycle arrest in HeLa cells. Methods HeLa cells were treated with 17-AAG. The cell cycle and chymotrypsin-like proteasome activity were detected by flow cytometry and fluorospectrophotometry respectively. Ubb siRNA was transfected into HeLa cells. Knockdown of Ubb was assayed by Real Time PCR. The expression of Cdkl and Hecl was tested by Western Blot. Results 17-AAG induced dose-dependent cell cycle arrest at G2/M phase and simultaneously increased chymotrypsin-like proteasome activity in HeLa cells. RNA interference of Ubb suppressed 17-AAG-induced cell cycle arrest. 17-AAG-induced cell cycle arrest was mediated by decrease of Cdkl and Hecl, which was inhibited by knockdown of Ubb. Conclusion 17-AAG-induced cell cycle arrest is mediated by ubiquitylation of cell-cycle checkpoint proteins, indicating Ubb as a possible target for 17-AAG-induced cell cycle arrest in HeLa cells.