Fbxw7对肝细胞癌肿瘤干细胞干性的影响研究

The effect of Fbxw7 on the stemness of hepatocellular carcinoma tumour stem cells

目的:观察Fbxw7对肝细胞癌(HCC)来源的肿瘤干细胞干性特性的影响,并探讨Fbxw7作为HCC肿瘤干细胞研究靶点的可行性。方法:实时荧光定量聚合酶联反应(qRT-PCR)及蛋白质印迹法(Western blot)检测HCC细胞中Fbxw7的表达水平。流式细胞仪检测HCC来源的CD133 +肿瘤干细胞细胞凋亡情况。裸鼠皮下成瘤实验检测Fbxw7在体内的增殖能力(使用6~8周龄的BALB-c雌性裸鼠6只,经简单随机抽样分成OE-NC组和OE-Fbxw7组,每组3只,皮下注射细胞后监测裸鼠中的肿瘤形成情况约36 d)。两组间比较采用 t检验,多组间比较采用单因素方差分析。 结果:HCC细胞系(SMMC-7721、HepG2、HCCLM3、HuH-7、Hep3B)中Fbxw7蛋白水平表达低于正常肝细胞(MIHA)(0.908±0.057、0.750±0.088、0.763±0.118、0.381±0.078、0.469±0.023比1.306±0.070, F=54.630, P均<0.01)。将Fbxw7过表达质粒转染到Hep 3b、HuH-7来源的CD133 +肿瘤干细胞中,Hep 3b细胞OE-Fbxw7组的凋亡率增加(18.157±2.003比8.197±1.398, t=7.061, P<0.01),HuH-7细胞OE-Fbxw7组的凋亡率增加(14.086±1.210比7.925±0.320, t=8.525, P<0.01)。裸鼠皮下成瘤模型中,Hep 3B来源CD133 +肿瘤干细胞OE-Fbxw7组肿瘤体积减小(66.076±28.848比788.342±85.831, t=13.820, P<0.01),Huh7来源CD133 +肿瘤干细胞OE-Fbxw7组肿瘤体积同样减小(69.194±46.569比545.423±101.865, t=7.364, P<0.01)。 结论:Fbxw7在肝细胞癌中低表达,过表达Fbxw7能抑制肝细胞癌肿瘤干细胞干性,影响成瘤情况。

Objective:To investigate the effect of Fbxw7 on the stemness properties of hepatocellular carcinoma(HCC)-derived tumour stem cells and to explore the feasibility of Fbxw7 as a target for HCC tumour stem cell research.Methods:The expression level of Fbxw7 in HCC cells was detected by real-time quantitative fluorescence polymerase chain reaction(qRT-PCR)and protein blotting(Western blotting).Flow cytometry was used to detect apoptosis in HCC-derived CD133+tumour stem cells.The in vivo proliferative capacity of Fbxw7 was examined in a subcutaneous tumour formation assay in nude mice(6 BALB-c female nude mice(purchased from Collective Pharmacology and Biotechnology),6 mice at 6-8 weeks of age were used,and were divided into the OE-NC group and the OE-Fbxw7 group,with 3 mice in each group,and the tumour formation was monitored for approximately 36 day after the subcutaneous injection of the cells).Comparisons between two groups were made by t-test,and comparisons between multiple groups were made by one-way analysis of variance(ANOVA).Results:The expression of Fbxw7 protein levels in HCC cell lines(SMMC-7721,HepG2,HCCLM3,HuH-7,Hep3B)was lower than that in normal hepatocytes(MIHA)(0.908±0.057,0.750±0.088,0.763±0.118,0.381±0.078,0.469±0.023 than 1.306±0.070,F=54.630,all P<0.01).Transfection of Fbxw7 overexpression plasmid into Hep 3b and HuH-7-derived CD133+tumour stem cells increased the apoptosis rate in the OE-Fbxw7 group of Hep 3b cells(18.157±2.003 vs.8.197±1.398,t=7.061,P,<0.01),and in the OE-Fbxw7 group of HuH7 cells(14.086±1.210 vs.7.925±0.320,t=8.525,P<0.01).In the nude mice subcutaneous tumourigenic model,tumour volume was reduced in the Hep 3B-derived CD133+tumour stem cells OE-Fbxw7 group(66.076±28.848 vs.788.342±85.831,t=13.820,P<0.01),and similarly in the Huh-7-derived CD133+tumour stem cells OE-Fbxw7 group(69.194±46.569 vs.545.423±101.865,t=7.364,P<0.01).Conclusion:Fbxw7 is lowly expressed in hepatocellular carcinoma,and overexpression of Fbxw7 inhibits the stemness of hepatocellular carcinoma tumour stem cells and affects tumour formation.