Skp2参与HeLa细胞G_2/M周期检查点的调控

具癌基因特性的Skp2在大多数肿瘤组织和肿瘤细胞中异常高表达,它作为SCFSkp2复合物的底物识别亚基调控p27KIP蛋白的稳定性而促进细胞G1/S期转换.为进一步明确Skp2与G2/M周期检查点的关系,在HeLa细胞中过表达Skp2以及通过反义寡核苷酸抑制Skp2表达.结果发现:Skp2能促进细胞周期运转,表现为S期细胞增多和G2/M期细胞减少,其中F-box结构域具有重要的功能意义;反义寡核苷酸抑制Skp2表达后,HeLa细胞发生显著的G2/M期阻滞;MTT检测结果表明,400nmol/L的Skp2的反义寡核苷酸能明显抑制HeLa细胞的增殖活性;Western印迹结果表明,HeLa细胞中Skp2可能通过负调控p21WAF的稳定性来参与G2/M检查点调控,这在用放线菌素D处理HeLa细胞的实验中得到验证.这些结果初步揭示了Skp2参与HeLa细胞G2/M周期检查点调控的分子机制.

Effect of Skp2 on G_2/M Checkpoint of HeLa Cells

Overexpression of oncogenic Skp2 has been observed in vaious types of human tumor tissues and cell lines. Skp2 positively regulates the G_1-S transition as a member of the F-box family of substrate-regulation subunits of SCF ubiquitin-protein ligase complexes. To determine the further relationship between Skp2 and the G_2/M checkpoint,ectopic expression of Skp2 or inhibition of Skp2 expression by antisense oligodeoxynucleotides was performed. Our results indicated that overexpression of Skp2 may stimulate progression of cell cycle of HeLa cells dependent of its F-box structure,characterized by increase in the S population and decrease in G_2/M population. Skp2-antisense treatment not only inhibits proliferation of HeLa cells as determined by MTT, but also induces G_2/M arrest determined by FCM. The result by Western blotting shows p21,an important G_2/M checkpoint regulator,invovled in cell cycle progression as an effector of Skp2. Furthermore,the inverse relationship between Skp2 and p21 was consistant with the result from HeLa cells treated with actinomycin D. This study will be helpful to reveal molecular mechanisms of Skp2 regulation G_2/M checkpoint in HeLa cells.