五味子乙素抑制视网膜母细胞瘤细胞生物学行为的分子机制研究

Inhibitory Effects and Molecular Mechanism of Schisandrin B in the Biologic Behaviors of Retinoblastoma Cells

目的:研究五味子乙素对人视网膜母细胞瘤Y79细胞增殖、凋亡、迁移及侵袭的影响,及其对共济失调毛细血管扩张突变基因(ATM)/转录因子E2F1信号通路的作用。方法:体外培养Y79细胞,ATM inhibitor质粒和ATM mimics质粒转染Y79细胞进行ATM沉默或过表达,细胞计数试剂盒-8(CCK-8)法测定细胞活力和增殖情况,流式细胞仪分析Y79细胞凋亡及周期分布,划痕实验检测Y79细胞迁移,Transwell实验分析细胞侵袭,Western Blotting实验检测Y79细胞中细胞周期蛋白(cyclin D1、cyclin B1和CDK2)、抑癌基因周期蛋白依赖性激酶抑制4b(INK4b)、INK4a、肿瘤抑制蛋白ARF、人体抑癌基因p53、人视网膜母细胞抑制蛋白pRB、ATM及E2F1蛋白表达。结果:五味子乙素呈浓度依赖性抑制Y79细胞活力、克隆、迁移及侵袭,将Y79细胞阻滞在G0/G1期,诱导其凋亡;另外,五味子乙素显著诱导INK4b、INK4a、ARF、p53、pRB、ATM及E2F1蛋白表达,显著抑制cyclin D1、cyclin B1及CDK2蛋白水平(P<0.05);ATM沉默能部分降低五味子乙素对Y79细胞生物学行为的抑制作用。结论:五味子乙素通过激活ATM/E2F1信号传导,进而抑制视网膜母细胞瘤细胞增殖,提示ATM/E2F1信号通路可能是视网膜母细胞瘤治疗的新靶标。

Objective:To study the effects of schisandrin B on the proliferation,apoptosis,migration and invasion of human retinoblastoma cells,and study its influence on ATM/E2F1 pathway.Methods:Y79 cell lines were cultured in vitro.ATM silence and overexpression were performed by transfecting with ATM inhibitor or mimics plasmid.The cell viability and proliferation were measured by CCK-8 assay.The cell apoptosis and cycle distribution were determined by flow cytometry.Linear scratch wounds were used to analyze the migration,and Transwell assay was used to observe the invasion of Y79 cells.Western blotting was used to detect the expression levels of cyclin D1,cyclin B1,CDK2,INK4b,INK4a,ARF,p53,pRB,ATM and E2F1.Results:Schisandrin B significantly inhibited the cell viability,colonies,migration and invasion of Y79 cells in a concentration-dependent manner.Schisandrin B greatly increased the numbers of Y79 cells in G0/G1 phase and induced the cell apoptosis.In addition,schisandrin B dramatically induced the expression levels of INK4b,INK4a,ARF,p53,pRB,ATM and E2F1,and significantly suppressed the expressions of cyclin D1,cyclin B1 and CDK2(P<0.05).ATM silence could partly decreased the inhibitory effects of schisandrin B on the biologic behaviors of Y79 cells.Conclusion:Schisandrin B reduces the proliferation of Y79 cells by decreasing ATM/E2F1 cascade,suggesting that the ATM/E2F1 axis may be a novel therapeutic target in the treatment of quercetin.