二甲双胍对肝癌PLC/PRF/5肿瘤干细胞增殖分化、凋亡、迁移的影响

目的:探究二甲双胍对肝癌PLC/PRF/5肿瘤干细胞(CSC)增殖分化、凋亡、迁移的影响。方法:体外特殊条件培养法培养PLC/PRF/5 CSC细胞,用不同浓度(5、10、20 mmol/L)二甲双胍处理PLC/PRF/5 CSC,以未进行处理的PLC/PRF/5 CSC为对照组,在显微镜下观察细胞形态;CCK-8法检测各组细胞活力;流式细胞术检测各组细胞凋亡率;划痕实验检测各组细胞迁移能力;蛋白免疫印迹法检测各组细胞蛋白表达情况;构建裸鼠肝癌移植瘤模型,检测二甲双胍对PLC/PRF/5 CSC成瘤能力的影响。结果:PLC/PRF/5 CSC具有成球能力,经二甲双胍处理后,PLC/PRF/5成球细胞数目显著减少;同一时间段,与对照组比较,二甲双胍处理组细胞活力显著降低,且呈剂量依赖性(P<0.05),随着处理时间的增加,各组细胞活力逐渐降低;与对照组比较,二甲双胍处理组细胞凋亡率、凋亡蛋白Caspase-3、Caspase-9、迁移蛋白E-cadherin的表达显著升高,细胞迁移率、细胞干性基因Oct4、Nanog蛋白表达、迁移蛋白N-cadherin的表达显著降低,且呈剂量依赖性(P<0.05);与对照组比较,随着接种时间的增加,二甲双胍处理组裸鼠肿瘤生长速度较慢(P<0.05),且随着二甲双胍浓度的增加,裸鼠肿瘤生长速度逐渐变慢。结论:二甲双胍能够抑制肝癌PLC/PRF/5 CSC的增殖和迁移,促进其凋亡,可能是靶向肝癌CSC治疗的潜在药物。

Effect of metformin on proliferation,differentiation,apoptosis and migration of cancer stem cells in liver cancer PLC/PRF/5

Objective:To investigate the effects of metformin on proliferation,differentiation,apoptosis and migration of liver PLC/PRF/5 tumor stem cells(CSC).Methods:PLC/PRF/5 CSC cells were cultured under special conditions in vitro,and were treated with different concentrations of metformin(5,10,20 mM),the untreated PLC/PRF/5 CSC was used as the control group.Cell morphology was observed under microscope;cell viability was detected by CCK-8 method;the apoptosis rate was detected by flow cytometry;the ability of cell migration was detected by scratch test;the protein expression of each group was detected by Western blot(WB);the effect of metformin on the tumorigenicity of PLC/PRF/5 stem cells was detected by xenograft tumor model of liver cancer in nude mice.Results:PLC/PRF/5 CSC had the ability of balling,and the number of PLC/PRF/5 CSC globular cells was significantly reduced after metformin treatment;at the same time,compared with that in the control group,the cell viability of metformin treatment group was significantly decreased,in a dose-dependent manner(P<0.05),with the increase of treatment time,the cell viability decreased gradually;compared with those in the control group,the apoptosis rate and the expression of Caspase-3,Caspase-9 and E-cadherin were significantly higher in metformin treatment group,the cell migration rate,the expression of Oct4,Nanog protein and N-cadherin were significantly lower,in a dose dependent manner(P<0.05);with the increase of inoculation time,compared with that in the control group,the growth rate of tumor in metformin treatment group was slower than that in control group(P<0.05),with the increase of metformin concentration,the growth rate of tumor in nude mice gradually slowed down.Conclusion:Metformin can inhibit the proliferation and migration of liver PLC/PRF/5 CSC and promote its apoptosis,and it may be a potential drug for CSC targeted therapy of HCC.