高表达Twist基因促进人乳腺癌细胞MCF-7干细胞样细胞富集和迁移

Overexpression of Twist gene promotes cancer stem-like cell enrichment and cell migration ability in MCF-7 human breast cancer cells

目的探讨人乳腺癌细胞系MCF-7中高表达Twist基因对MCF-7干细胞特性细胞的影响。方法构建重组质粒pBABE-puro-myc-Twist,HEK293T细胞包装逆转录病毒,并感染MCF-7细胞,通过筛选获得成功转入Twist的MCF-7/Twist细胞和MCF-7/Vector对照细胞,再通过长程无血清悬浮培养方法富集具有干细胞特性的乳腺细胞小球(Mammosphere),并通过Western blot法检测myc-Twist及Twist诱导后富集的Mammosphere相关CSC蛋白标记的表达,Transwell法检测细胞的迁移能力。结果成功构建逆转录病毒质粒pBABE-puro-myc-Twist;Twist基因在靶细胞内稳定表达;长程无血清悬浮培养富集出具有干细胞特性的Mammosphere;MCF-7/Twist细胞肿瘤干细胞样细胞富集能力增强(P<0.05);myc-Twist、Twist及SOX2、OCT4蛋白在MCF-7/Twist Mammosphere细胞中表达上调;MCF-7/Twist Mammosphere细胞迁移能力增加(P<0.05)。结论 Twist促进MCF-7肿瘤干细胞样细胞富集能力增强,并促进其迁移。

Objective To overexpress Twist gene in MCF-7 human breast cancer cells and investigate its effect on cancer stem-like cell（ CSC） in MCF-7.Methods Recombinant plasmid pBABE-puro-myc-Twist was constructed and retrovirus was packaged in HEK293 T cells.MCF-7 cells were transfected with the retrovirus carrying Twist gene or the controls.MCF-7 /Twist cells and MCF-7 /Vector control cells were selected.Mammospheres with characteristics of stem-like cells were collected by long term serum-free suspension cultivation.The expressions of myc-Twist and CSC marker proteins induced by Twist in MCF-7 /Twist mammospheres and MCF-7 /Vector mammospheres were determined by qRT-PCR and Western blotting.Cell migration was analyzed by Transwell assay.Results Twist gene was efficiently delivered into MCF-7cells,resulting in the stable expression of Twist mRNA.Mammospheres with characteristics of stem-like cells were collected by long term serum-free suspension cultivation.CSC enrichment was enhanced notably in MCF-7 / Twist cells（ P〈0.05）.The expressions of myc-Twist,Twist and CSC marker proteins SOX2 and OCT4 were upregulated in MCF-7 /Twist mammospheres and cell migration was enhanced（ P〈0.05）.Conclusion Twist plays an important role in enhancing CSC enrichment and promoting cell migration in MCF-7 cells.