Bmi-1小分子抑制剂PTC-209对肾癌细胞生物学行为的影响及机制

Influence and mechanism of Bmi-1 small-molecule inhibitor PTC-209 on biological behavior of renal carcinoma

目的观察Bmi-1小分子抑制剂PTC-209对肾细胞癌(简称肾癌)细胞生物学行为的影响,并探讨其机制。方法以0、1、2. 5、5、10μmol/L的PTC-209作用于肾癌786-O细胞,采用CCK-8法测算培养24、48 h时的细胞存活率,流式细胞术检测细胞凋亡及细胞周期分布情况,划痕实验观察细胞迁移能力,克隆形成实验观察细胞克隆集落形成能力,荧光定量PCR法及Western blotting法检测细胞中的Notch1及Notch信号通路下游靶基因发状分裂相关增强子1(HES1)、髓细胞组织增生蛋白(MYC)、细胞周期蛋白依赖性激酶抑制因子2A(CDKN2A)、Cyclin D1和Bcl-2。结果随着PTC-209作用浓度升高及作用时间的延长,786-O细胞存活率减少(P均<0. 05);随着PTC-209作用浓度升高,786-O细胞凋亡率、G_0/G_1期细胞比例增加,S期及G_2/M期细胞比例、细胞迁移率及克隆形成数减少(P均<0. 05);与0μmol/L比较,5μmol/L的PTC-209作用后786-O细胞中Notch1、HES1、MYC、CDKN2A、Cyclin D1、Bcl-2基因及蛋白表达均减少(P均<0. 05)。结论 PTC-209可抑制肾癌细胞增殖,促进细胞凋亡,将细胞阻滞于G_0/G_1期,减弱细胞迁移及克隆形成能力;该作用与PTC-209影响肾癌细胞中Notch信号通路相关基因表达有关。

Objective To explore the influence and mechanism of Bmi-1 small-molecule inhibitor PTC-209 on the biological behavior of renal cell carcinoma (renal carcinoma). Methods The 786-O cells were treated with 0,1,2.5,5 and 10 μmol/L PTC-209 for 24 and 48 h.The effect of PTC-209 on 786-O cell proliferation was assessed by CCK8.Flow cytometry was applied to examine the effect of PTC-209 on 786-O cell cycle and apoptosis.Wound healing assay and colony forming assay were applied to evaluate the capacity of cell migration and colony formation,respectively.Quantitative real-time PCR and Western blotting were applied to evaluate the mRNA and protein expression of NOTCH1,HES1,MYC,CDKN2A,CyclinD1 and Bcl-2. Results PTC-209 gradually inhibited the growth of 786-O cells with increase of drug doses and action time ( P <0.05).PTC-209 induced both apoptosis and cell cycle arrest of 786-O cells in a dose-dependent manner.The percentages of cells in the G0/G1 phase increased while in the S phase and G 2/M phase decreased.The migration and colony forming ability were also inhibited by PTC-209 in a dose-dependent manner ( P <0.05).Comparing with 0 μmol/L PTC-209,5 μmol/L PTC-209 decreased the gene and protein expression of NOTCH1,HES1,MYC,CDKN2A,CyclinD1 and Bcl-2 (all P <0.05). Conclusion PTC-209 inhibits 786-O cell proliferation,induces apoptosis,blocks the cells in the G 0/G 1 phase,and significantly suppresses tumor cell migration and colony formation,and its mechanism may be related to NOTCH signaling pathway-related gene expression.