川陈皮素调节CDK1/CCNB1/PLK1信号通路对肝癌细胞恶性生物学行为的影响

Effect of Nobiletin on the malignant biological behaviors of liver cancer cells by regulating the CDK1/CCNB1/PLK1 signaling pathway

目的 探究川陈皮素(NOB)调节细胞周期蛋白依赖性激酶1(CDK1)/细胞周期蛋白B1(CCNB1)/Polo样激酶1(PLK1)信号通路对肝癌细胞恶性生物学行为的影响。方法 用浓度为10.0~160.0μmol/L的川陈皮素处理人肝癌细胞(Huh-7),CCK-8法检测细胞活性,筛选最佳川陈皮素浓度;将Huh-7细胞分为对照组(Control组),NOB低(L组)、中(M组)、高(H组)浓度组、CDK1/CCNB1抑制剂组(CE组);用平板克隆法、流式细胞仪、划痕实验、Transwell实验分别检测细胞增殖、凋亡、迁移、侵袭;免疫印迹法(Western blot)检测细胞增殖相关蛋白(Ki67、Cyclin D1)、凋亡相关蛋白(Bax、Caspase-3)、迁移侵袭相关蛋白(MMP-2、MMP-9)、CDK1、CCNB1、PLK1蛋白表达;用裸鼠移植瘤方法检测NOB对肝癌移植瘤生长的影响。结果 选择20μmol/L、40μmol/L、80μmol/L的NOB浓度用于后续实验。与Control组比较,L、M、H、CE组集落形成数、细胞划痕愈合率、细胞侵袭数量及Ki67、Cyclin D1、MMP-2、MMP-9、CDK1、CCNB1、PLK1表达水平降低,细胞凋亡率、Caspase-3、Bax表达水平明显上升(P<0.05)。裸鼠成瘤实验发现,NOB组移植瘤生长更为缓慢,移植瘤的质量和体积明显下降,且CDK1、CCNB1、PLK1表达下调(P<0.05)。结论 NOB抑制CDK1/CCNB1/PLK1信号通路抑制肝癌细胞的恶性生物学行为。

Objective To investigate the effect of Nobiletin(NOB)on the malignant biological behaviors of liver cancer cells by regulating the cyclin dependent kinase 1(CDK1)/cyclin B1(CCNB1)/polo like kinase 1(PLK1)signaling pathway.Methods Human liver cancer cells(Huh-7)were treated with NOB at a concentration of 10.0-160.0μmol/L,followed by the screening of the optimal concentration by cell counting kit-8(CCK-8)assay.Huh-7 cells were treated with blank control(Control group),low-dose NOB(L group),medium-dose NOB(M group),high-dose NOB(H group),and CDK1/CCNB1 inhibitor(CE group).Cell proliferation,apoptosis,migration,and invasion were examined by colony formation assay,flow cytometry,wound healing assay and Transwell assay respectively.Western blot was applied to detect the protein expressions of proliferation-related proteins(Ki67,Cyclin D1),apoptosis-related proteins(Bax,Caspase-3),migration/invasion-related proteins(MMP-2,MMP-9),CDK1,CCNB1,and PLK1.A nude mouse xenograft model was created to detect the in vivo effect of NOB on the growth of liver cancer.Results NOB concentrations of 20μmol/L,40μmol/L,and 80μmol/L were selected as the low-dose,medium-dose and high-dose treatment for subsequent experiments,respectively.Compared with those of the Control group,cells in the L,M,H,and CE groups showed significantly lower number of colonies,wound healing rate,invasive cell number,protein levels of Ki67,Cyclin D1,MMP-2,MMP-9,CDK1,CCNB1,and PLK1,and higher apoptotic rate and protein levels of Caspase-3,and Bax(P<0.05).In the nude mouse xenograft model,NOB treatment yielded a significantly slower tumor growth,lower tumor weight and volume and lower protein levels of CDK1,CCNB1,and PLK1(P<0.05).Conclusion NOB inhibits the malignant biological behavior of liver cancer cells by inactivating the CDK1/CCNB1/PLK1 signaling pathway.