腺病毒靶向干扰S期激酶相关蛋白2基因对膀胱癌细胞生物学行为的影响

Effect on biological behavior of bladder cancer cells by adenovirius-mediated gene silencing of S-phase kinase-associated protein 2

目的 观察沉默S期激酶相关蛋白2（Skp2）基因对细胞周期蛋白依赖性激酶抑制蛋白27（P27KiP1）、第10号染色体缺失的磷酸酶及张力蛋白同源物基因（PTEN）的表达调控及对膀胱癌细胞生长活性的影响,探讨其在肿瘤侵袭、凋亡中的作用机制.方法 采用Western blot法检测经腺病毒载体（Ad-Skp2）转染后膀胱癌细胞株EJ的Skp2、P27kip1和PTEN的表达改变;用细胞划痕实验、Boyden小室体外侵袭实验、吖啶橙/溴化乙锭（AO/EB）荧光染色法、噻唑蓝（MTT）比色法反映转染后细胞迁移力、外侵袭力、凋亡及生长活性的变化.结果 转染后48 h的膀胱癌EJ细胞,Skp2蛋白表达开始下降,以转染后96 h最明显;而P27kiP1和PTEN在转染96 h后,表达显著增加（P＜0.05）;转染后的EJ细胞体外侵袭力下降[（11.7±1.2）％比（69.5±1.4）％,P＜0.05],迁移能力减弱（P＜0.05）,细胞凋亡增多（11.8％比45.6％,P＜0.05）,生长活性降低（P＜0.05）.结论 膀胱癌细胞中的Skp2表达下调可能通过负向调控P27kipl、PTEN而影响癌细胞的分裂周期,从而使癌细胞衰老,最终诱导凋亡.

Objective To investigate the regulatory effect of cyclin dependent kinase inhibitor protein 27 （P27KiP1）,phosphatase and tensin homolog deleted on chromosome ten （PTEN） expression and cell viability by silencing human gene S-phase kinase-associated protein 2 （Skp2）,and further reseach for its role on invasion and apoptosis of cancer.Methods Adenoviral shRNA vector encoding Skp2 was transfected into EJ cell line.The expression of Skp2,P27kip1 and PTEN were detected by Western blotting assay; the metastatic potential and apoptosis were examined by in vitro cell wound model,Boyden chamber invasion assay and acridine orange-ethidium bromide fluorescent staining.Results The expression of Skp2 was decreased in the EJ cells after transfected with Skp2 shRNA,and the expression of P27kip1,PTEN were significantly up-regulated （P 〈 0.05） ; the motility,invation of EJ cells [（11.7 ± 1.2） % vs.（69.5 ±1.4） %] were inhibited and apoptosis （11.8% vs.45.6%） was enhanced.Conclusion The silencing of Skp2 may take an important role in cell cycle of bladder cancer cells by regulating P27kip1 and PTEN,which resulted in cell aging and lastly death.