沉默细胞骨架相关蛋白2抑制肝癌细胞增殖和迁移并促进细胞凋亡

Silence of cytoskeleton-associated protein 2 represses cell proliferation and migration and promotes apoptosis in liver cancer cell lines

目的:研究细胞骨架相关蛋白2(cytoskeleton-associated protein 2,CKAP2)对肝癌细胞的增殖、凋亡和迁移的作用及其机制。方法:培养正常肝细胞L02和肝癌细胞系HepG2,Huh7和SMMC-7721。采用实时PCR和蛋白质印迹法检测CKAP2的表达水平。进一步将HepG2细胞分为对照(Control)组、阴性对照(NC)组和沉默CKAP2(siCKAP2)组。NC组和siCKAP2组细胞分别转染siControl和siCKAP2。采用CKK-8法检测细胞活性,BrdU掺入法检测细胞增殖,凋亡试剂盒检测细胞凋亡,transwell实验检测细胞的迁移和侵袭。采用蛋白质印迹法检测cleavedcaspase 3,Bax,E-cadherin,N-cadherin,Vimentin以及磷酸化的Janus激酶2(Janus kinase 2,JAK2)和磷酸化的信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)的蛋白质表达水平。结果:与正常肝细胞L02比较,CKAP2在肝癌细胞系HepG2,Huh7和SMMC-7721中的表达显著上调(均P<0.05)。与NC组比较,siCKAP2组细胞活性和增殖率显著下降(均P<0.05);细胞凋亡率升高,cleaved-caspase 3和Bax的蛋白质表达水平显著上调(均P<0.05);细胞迁移和侵袭显著减少(均P<0.05);E-cadherin蛋白质表达水平显著上调,Vimentin,Ncadherin,磷酸化的JAK2和磷酸化的STAT3的蛋白质表达水平显著下调(均P<0.05)。结论:沉默CKAP2基因抑制肝癌细胞的增殖、迁移和侵袭,促进其凋亡,JAK2/STAT3信号通路可能参与这些过程。

Objective:To investigate the roles of cytoskeleton-associated protein 2(CKAP2)in proliferation,apoptosis,and migration in liver cancer cells and the potential mechanisms.Methods:Human normal hepatocyte L02 and liver cancer cell lines HepG2,Huh7,and SMMC-7721 were cultured.The CKAP2 expression was detected by real-time PCR and Western blotting.HepG2 cells were randomly divided into a control group,a negative control(NC)group,and a CKAP2 silencing(siCKAP2)group.CCK-8 and BrdU assays were used to evaluate cell viability and proliferation,respectively.Transwell assay was employed to determine cell migration and invasion.The protein levels of cleaved-caspase 3,Bax,E-cadherin,N-cadherin,Vimentin,phosphorylated Janus kinase 2(p-JAK2),and phosphorylated signal transducer and activator of transcription 3(p-STAT3)were determined byWestern blotting.Results:Compared with normal hepatocyte L02,CKAP2 was highly expressed in liver cancer cell lines HepG2,Huh7,and SMMC-7721(all P<0.05).Compared with the NC group,cell viability and proliferation rate of the siCKAP2 group were decreased(both P<0.05).The apoptotic rate,protein expression of cleaved-caspase 3 and Bax in the siCKAP2 group were significantly higher than those in the NC group(all P<0.05).Compared with the NC group,cell migration and invasion rates of the siCKAP2 group were significantly attenuated(both P<0.05).Compared with the NC group,E-cadherin protein expression in siCKAP2 group was increased,while protein expression levels of Vimentin,N-cadherin,p-JAK2,and p-STAT3 were decreased(all P<0.05).Conclusion:CKAP2 gene silence inhibits proliferation,migration,and invasion,and promotes apoptosis in liver cancer cells,while JAK2/STAT3 signaling pathway may be involved in these processes.