过表达Bmi-1促进CD133^(+)CD44^(+)HCT116结肠癌干细胞发生上皮间质转化

Overexpression of Bmi-1 promotes epithelial-mesenchymal transition in CD133^(+)CD44^(+)HCT116 colon cancer stem cells

目的探讨Bmi-1促进结肠癌干细胞发生迁移、侵袭的作用及机制。方法常规培养结肠癌HCT116细胞,成球培养基法(SFM)和磁珠分选法(MACS)富集和筛选CD133^(+)CD44^(+)HCT116结肠癌干细胞;流式细胞学鉴定结肠癌干细胞CD133和CD44表型,体外平板克隆实验和CCK8法鉴定细胞增殖克隆能力,裸鼠体内成瘤实验鉴定细胞致瘤能力。构建过表达Bmi-1质粒并转染CD133^(+)CD44^(+)HCT116结肠癌干细胞,Western blotting验证细胞中Bmi-1、E-cadherin及Vimentin的表达情况。平板划痕实验和Transwell实验评价转染后不同细胞的迁移和侵袭能力。结果CD133^(+)CD44^(+)HCT116结肠癌细胞具有更强的体外克隆增殖能力和体内致瘤能力,可作为结肠癌干细胞研究模型;过表达Bmi-1后促进CD133^(+)CD44^(+)HCT116结肠癌干细胞的E-cadherin表达下降,而Vimentin表达上升;过表达Bmi-1后细胞24 h迁移速度为(18.44±0.59)μm/h,明显高于对照组(1.88±0.21)μm/h和普通组(1.92±0.36)μm/h(P<0.05),同时其侵袭细胞数(37.67±2.51)也明显高于对照组(9.33±0.58)和普通组(7.67±0.58)(P<0.05)。结论Bmi-1介导CD133^(+)CD44^(+)HCT116结肠癌干细胞发生上皮间质转化(EMT)而促进肿瘤侵袭、转移。

Objective To investigate the role and mechanism by which Bmi-1 may promote invasion and migration of CD133^(+)CD44^(+)HCT116 colon cancer stem cells(CCSCs).Methods Colon cancer cell line HCT116 was normally cultured,and CD133^(+)CD44^(+)HCT116 CCSCs were enriched and screened by sphere formation media(SFM)and magneticactivated cell sorting(MACS).The expression of CD133 and CD44 in CCSCs was assessed by flow cytometry;colony formation assay,cell proliferation assay and viability assay using CCK8,and transplantation assay using nude mice injected with CCSCs were used to examine the CCSCs.Bmi-1 was overexpressed in CD133^(+)CD44^(+)HCT116 cells by transfection with the Bmi-1/pcDNA3.1(^(+))expression plasmid and confirmed by Western blotting.The expression of E-cadherin and Vimentin in CCSCs was also tested by Western blotting.The invasion and migration abilities of CCSCs after transfection were measured by Transwell invasion and wound healing assays,respectively.Results The CD133^(+)CD44^(+)HCT116 cells exhibited greater cloning efficiency,an enhanced proliferative ability,increased cell viability and stronger tumorigenicity;these cells were used as the CCSCs for subsequent experiments.Overexpression of Bmi-1 in CD133^(+)CD44^(+)HCT116 cells resulted in the downregulation of E-cadherin and upregulation of Vimentin at the protein level.The migration rate of CD133^(+)CD44^(+)HCT116 cells[(18.44±0.59)μm/h]was increased in the Bmi-1/pcDNA3.1(^(+))-transfected group,more than control group[(1.88±0.21)μm/h]and normal group[(1.92±0.36)μm/h].The number of invasion cell(37.67±2.51)was more than control group(9.33±0.58)and normal group(7.67±0.58)(P<0.05).Conclusion Bmi-1 promotes invasion and migration of CD133^(+)CD44^(+)HCT116 CCSCs through inducing epithelial-mesenchymal transition(EMT).