Hedgehog信号通路调控Twist高表达乳腺肿瘤干细胞样细胞的富集及迁移  

Hedgehog signaling pathway regulates the enrichment and migration of Twist highly expressed breast cancer stem cell-like cells

作　　者：周明莉[1,2] 唐石伏[3] 张海龙 柳满然[2] 

机构地区：[1]成都市第三人民医院检验科,四川成都610031 [2]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016 [3]广西中医药大学第三附属医院检验科,广西壮族自治区柳州545001 [4]安顺市人民医院检验科,贵州安顺561000

出　　处：《肿瘤》2017年第10期1024-1031,1046,共9页Tumor

基　　金：国家自然科学基金面上项目(编号:31171336);国家自然科学基金地区科学基金项目(编号:81560430)~~

摘　　要：目的 :探讨阻断Hedgehog信号通路对Twist高表达乳腺癌干细胞样细胞微球体富集及其迁移的影响。方法:分别采用蛋白质印迹法和微球体细胞形成实验检测正常乳腺上皮MCF-10A细胞、乳腺癌MCF-7和HS578T细胞中Twist蛋白的表达水平及微球体细胞形成能力。蛋白质印迹法检测MCF-10A、MCF-7和HS578T微球体细胞中Twist、GLI1家族锌指蛋白(GLI family zincnger1,GLI1)和音猬因子(Sonic Hedgehog,SHh)蛋白的表达情况。采用不同浓度的Hedgehog信号通路特异性抑制剂GDC-0449(10、20和30μmol/L)处理Twist蛋白表达水平最高的乳腺癌HS578T微球体细胞96 h,分别采用微球体细胞形成实验和Transwell小室法检测GDC-0449对HS578T微球体细胞再成球能力和迁移能力的影响,并进一步采用蛋白质印迹法检测HS578T微球体细胞中GLI1、性别决定区Y框蛋白2(sex determining region Y-box 2,SOX2)和八聚体绑定转录因子4(octamer-binding transcription factor 4,OCT4)蛋白的表达情况。结果:乳腺癌HS578T细胞中Twist蛋白的表达水平明显高于MCF-10A和MCF-7细胞(P值均<0.05),且具有最强的微球体形成能力(P值均<0.05)。HS578T微球体细胞中Twist、GLI1和SHh蛋白水平均明显高于MCF-10A和MCF-7微球体细胞(P值均<0.05);经抑制剂GDC-0449处理后,乳腺癌HS578T微球体细胞的成球能力及迁移能力均明显降低(P值均<0.05),GLI1、SOX2和OCT4蛋白的表达水平明显下调(P值均<0.05)。结论:阻断Hedgehog信号通路的活化可抑制Twist高表达乳腺癌细胞微球体的形成及迁移能力。Objective： To investigate the effects of Hedgehog signaling pathway on the enrichment and migration of Twist highly expressed breast cancer stem cell-like mammospheres.Methods： The expression level of Twist protein in normal breast epithelial MCF-10A cells and breast cancer MCF-7 and HS578T cells was detected by Western blotting.The mammosphere formation efficiency of MCF-10A, MCF-7 and HS578T cells was detected by microsphere formation assay, respectively. Then the expression levels of Twist, GLI family zinc finger 1 （GLI1） and Sonic Hedgehog （SHh） proteins in mammosphere cells derived from MCF-10A, MCF-7 and HS578T cells were detected by Western blotting. The HS578T mammosphere cells with the highest expression level of Twist protein were selected, and treated with the different concentrations （10, 20 and 30 ~mol/L） of Hedgehog signaling pathway inhibitor GDC-0449. The mammosphere reformation efficiency and migration ability of HS578T mammosphere cells were detected by microsphere formation assay and Transwell assay, respectively. The expression levels of GLI1, sex determining region Y-box 2 （SOX2） and octamer-binding transcription factor 4 （OCT4） in HS578T mammosphere cells after treatment with GDC-0449 were detected by Western blotting. Results： The expression level of Twist protein in breast cancer HS578T cells was significantly higher than that in MCF-10A and MCF-7 cells （both P 〈 0.05）, and the mammosphere formation efficiency of HS578T cells was strongest （both P 〈 0.05）. The expression levels of Twist, GLI1 and SHh proteins in HS578T mammosphere cells were higher than those in MCF-10A and MCF-7 mammosphere cells （all P 〈 0.05）. After the treatment with Hedgehog signaling pathway inhibitor GDC-0449, the abilities of mammosphere formation and migration of HS578T mammosphere cells were significantly inhibited （both P 〈 0.05）. The expression levels of GLI1, SOX2 and OCT4 in HS578T mammosphere cells after treatment with GDC-0449 were significantly down-regulat

关 键 词：乳腺肿瘤 肿瘤干细胞 TWIST转录因子 Hedgehog信号通路 细胞运动 

分 类 号：R737.9[医药卫生—肿瘤]