摘要
目的观察细胞分裂周期蛋白6(cell division cycle 6,CDC6)在肝癌干细胞(HepG2-CSCs)中的表达,并探讨CDC6 siRNA对HepG2-CSCs增殖和迁移的影响。方法通过免疫磁珠分选法从肝癌细胞株HepG2中分离肝癌干细胞HepG2-CSCs,并通过流式细胞术鉴定细胞免疫表型,Western blot法检测HepG2-CSCs中CDC6蛋白的表达水平。将HepG2-CSCs分为两组,分别为对照siRNA组和CDC6 siRNA组,其中CDC6 siRNA组和对照siRNA组分别为通过lipofectamine 2000转染CDC6 siRNA和对照siRNA的HepG2-CSCs细胞,平板克隆形成实验和Transwell法分别检测CDC6 siRNA对HepG2-CSCs克隆形成和迁移的影响;Western blot检测CDC6 siRNA对HepG2-CSCs CDC6、wnt3a和β-catenin蛋白表达的影响。结果流式细胞术检测结果显示,CD133在HepG2和HepG2-CSCs中阳性率分别为(0.54±0.16)%和(98.82±0.95)%;Western blot结果显示CDC6蛋白在HepG2-CSCs细胞中的表达显著高于HepG2细胞(P<0.01);克隆形成实验结果显示,与对照siRNA组比较,CDC6 siRNA组细胞克隆形成率显著降低(P<0.01);Transwell检测结果显示,与对照siRNA组比较,CDC6 siRNA组HepG2-CSCs细胞迁移能力显著降低(P<0.01);Western blot结果显示,与对照siRNA组比较,CDC6 siRNA组CDC6、wnt3a和β-catenin蛋白表达显著降低(P<0.01)。结论CDC6在HepG2-CSCs中高表达,CDC6siRNA能够抑制wnt3a和β-catenin蛋白表达,抑制HepG2-CSCs克隆形成和迁移。
Objective To study the expression of cell division cycle 6(CDC6)in hepatocellular cancer stem cells(HepG2-CSCs)and investigate the effect of CDC6 siRNA on proliferation and migration of HepG2-CSCs.Methods HepG2-CSCs were isolated from HepG2 cell line by immunomagnetic bead sorting,and the cellular immune-phenotype was identified by flow cytometry.The expression of CDC6 protein in HepG2-CSCs was detected by Western blot.HepG2-CSCs were divided into two groups:control siRNA group and CDC6 siRNA group.HepG2-CSCs cells were transfected with CDC6 siRNA and control siRNA by lipofectamine 2000 in CDC6 siRNA group and control siRNA group,respectively.The effects of CDC6 siRNA on the clonal formation and migration of HepG2-CSCs were detected by plate cloning assay and Transwell method.Western blot was used to detect the expression of CDC6,wnt3a andβ-catenin proteins in HepG2-CSCs transfected with CDC6 siRNA.Results The results of flow cytometry showed that the positive rates of CD133 were(0.54±0.16)%and(98.82±0.95)%in HepG2 and HepG2-CSCs,respectively.Western blot results showed that the expression of CDC6 protein in HepG2-CSCs was significantly higher than that in HepG2(P<0.01).The results of plate cloning assay showed that the clone formation rate of HepG2-CSCs in CDC6 siRNA group was significantly lower than that in control-siRNA group.The results of Transwell showed that the number of cell migration in CDC6 siRNA group was significantly lower than that of control-siRNA group(P<0.01).Western blot results showed that the expression of CDC6,wnt3a andβ-catenin protein in CDC6 siRNA group was significantly lower than that in control-siRNA group(P<0.01).Conclusion CDC6 is highly expressed in HepG2-CSCs,and the siRNA interference with CDC6 can inhibit the expression of wnt3a andβ-catenin protein,and inhibit the clone formation and migration of HepG2-CSCs.
作者
牛广旭
郭晓娟
杨建华
刘小慧
NIU Guangxu;GUO Xiaojuan;YANG Jianhua;LIU Xiaohui(Department of Pathology,Handan Central Hospital,Handan 056001,China;Department of General Surgery,Handan Central Hospital)
出处
《山西医科大学学报》
CAS
2019年第12期1649-1654,共6页
Journal of Shanxi Medical University
基金
河北省青年科技项目指令性课题资助(20170227)
关键词
肝癌干细胞
CDC6
细胞增殖
细胞迁移
liver cancer stem cell
CDC6
cell proliferation
cell migration
