叉头框转录因子M1对人肝癌细胞增殖和凋亡及侵袭的影响

Effects of forkhead box M1 on proliferation, apoptosis and invasion in human hepatoma cells

目的探讨叉头框转录因子M1(FoxM1)对人肝癌细胞增殖、凋亡、侵袭的影响。方法应用定量聚合酶链反应及蛋白质印迹法检测6种常见人肝癌细胞系(HepG2、Huh-7、Hep3B、SMMC7721、QSG7701、BEL7402)及人正常肝细胞系LO-2中FoxM1的mRNA和蛋白表达水平。选取FoxM1高表达的肝癌细胞系,应用靶向FoxM1的短发卡RNA(shRNA)下调FoxM1表达,观察其对肝癌细胞凋亡、细胞周期、细胞增殖、迁移、侵袭能力的影响。观察下调FoxM1表达的肝癌细胞在裸鼠体内的成瘤情况。结果在6种常见肝癌细胞系及人正常肝细胞系中,HepG2及BEL7402的FoxM1的mRNA和蛋白表达水平最高,选取HepG2与BEL7402细胞系进行后续实验。应用靶向FoxM1的shRNA下调FoxM1的表达后,HepG2与BEL7402细胞系的shRNA组细胞凋亡率均高于对照组[(12.6±1.9)%比(4.6±0.5)%、(10.3±0.5)%比(3.9±1.9)%],G_(2)/M期细胞比例均低于对照组,细胞计数试剂盒-8实验96 h时450 nm处吸光度值均低于对照组,划痕实验中划痕愈合率均低于对照组,Transwell实验中穿膜细胞数均低于对照组(均P<0.05)。在动物体内实验中,HepG2细胞系shRNA组实验16、20 d肿瘤体积小于对照组,BEL7402细胞系shRNA组实验12、16、20 d肿瘤体积小于对照组,两细胞系shRNA组实验20 d时肿瘤质量均低于对照组(均P<0.05)。结论下调FoxM1能够促进肝癌细胞的凋亡,导致细胞周期发生停滞,抑制细胞增殖,降低细胞迁移、侵袭能力和体内成瘤能力。

Objective To investigate the effects of forkhead box M1(FoxM1) on proliferation, apotosis and invasion of human hepatoma cells. Methods The expression of FoxM1 in 6 kinds of common cell lines(HepG2, Huh-7, Hep3 B, SMMC7721, QSG7701, BEL7402) of human hepatoma and normal liver cell line LO-2 was detected by quantitative polymerase chain reaction and western blotting. Short hairpin RNA(shRNA) was transfected into the cell lines with higher FoxM1 expression level to down-regulate the expression of FoxM1. Apoptosis, cell cycle, proliferation, migration and invasion ability were studied. Tumor formation capacity in nude mice was assessed by down-regulation of FoxM1. Results Among 6 common liver cancer cell lines and human normal liver cell lines, the mRNA and protein expression levels of FoxM1 in HepG2 and BEL7402 were the highest. HepG2 and BEL7402 cell line were selected for follow-up experiments. After down-regulating the expression of FoxM1 by shRNA targeting FoxM1, the apoptosis rate of shRNA group in HepG2 and BEL7402 cell lines was higher than that of the control group[(12.6±1.9)% vs(4.6±0.5)%,(10.3±0.5)% vs(3.9±1.9)%], the proportion of cells in G_(2)/M phase was lower than that of the control group, and the absorbance value at 450 nm at 96 h in cell counting kit-8 experiment was lower than that of the control group, the wound healing rate in the scratch experiment was lower than that of the control group, and the number of transmembrane cells in the Transwell experiment was lower than that of the control group(all P<0.05). In vivo experiments, the tumor volume of HepG2 cell line shRNA group was less than that of the control group at 16 and 20 d;the tumor volume of BEL7402 cell line shRNA group was less than that of the control group at 12, 16 and 20 d, and the tumor quality of the two cell lines shRNA groups was lower than that of the control group at 20 d(all P<0.05). Conclusion Down-regulation of FoxM1 in hepatoma cell lines apoptosis is promoted, cell cycle arrest, the capability of cell growth, cell migration and invasion, and tumorigenesis in vivo all decrease.