[Effect of forced E-cadherin expression on adhesion and proliferation of human breast carcinoma cells].
[Effect of forced E-cadherin expression on adhesion and proliferation of human breast carcinoma cells].
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DOI：
10.3760/cma.j.issn.0529-5807.2010.12.011
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发表时间：
2010-12
期刊：
影响因子：
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通讯作者：
Li-juan Yang;Yu-qin Liu;B. Gu;X. Bian;H. Feng;Zhen-li Yang;Yanyouhong Liu
中科院分区：
文献类型：
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作者：
Li-juan Yang;Yu-qin Liu;B. Gu;X. Bian;H. Feng;Zhen-li Yang;Yanyouhong Liu
目的探讨上皮型钙粘蛋白（E-cad）在乳腺癌细胞粘附和增殖中的作用。方法将E-cad表达载体转染E-cad阴性的人乳腺癌MDA-MB-231细胞。G418筛选阳性克隆。Western blot检测E-cad、β-catenin（β-cat）和cyclin D1的表达。检测其细胞-细胞及细胞-基质粘附能力。E-cad/β-catenin相互作用通过免疫沉淀证实。MTT法检测细胞增殖;流式细胞仪检测细胞凋亡。结果建立了E-cad（+）细胞株Ecad-231-7和Ecad-231-9。当在超低结合培养皿中培养时，Ecad-231细胞在悬浮液中生长，而Ecad-231-7和Ecad-231-9细胞在大夹具中生长。当与HCT 116细胞共培养时，MDA-MB-231、Ecad-231-7和Ecad-231-9在30 min时的平均粘附率分别为39.0%、60.0%和59.5%。EDTA作用5 min的平均脱附率分别为37.4%、4.2%和7.4%。E-cad的表达增强了血型和异型细胞间的粘附和细胞与基质的粘附。Western blot结果显示，外源性强表达的E-cad可与内源性β-cat联合收割机结合，而下游cyclin D1表达明显降低。MDA-MB-231、Ecad-231-7和Ecad-231-9的细胞凋亡率分别为1.8%、2.0%和2.1%。E-cad表达对常规培养的肿瘤细胞凋亡无明显影响。β-cat在胞浆中表达增加。结论成功建立了稳定表达E-cad的单克隆肿瘤细胞株Ecad-231-7和Ecad-231-9。E-cad可通过β-cat和cyclin D1途径增强人乳腺癌细胞的粘附，抑制其增殖。
OBJECTIVE To investigate the role that E-cadherin (E-cad) plays on cell adhesion and proliferation of human breast carcinoma. METHODS E-cad expression vector was transfected into an E-cad-negative human breast carcinoma MDA-MB-231 cells. G418 was used to screen positive clones. E-cad, β-catenin (β-cat) and cyclin D1 expressions of these clones were confirmed by Western blot. Their cell-cell and cell-matrix adhesion abilities were detected. E-cad/β-catenin interaction was confirmed by immunoprecipitation. Cell proliferation was evaluated by MTT. Cell apoptosis was analyzed by flow cytometry. Direct two-step immunocytochemistry was used to detect the localization of β-cat. RESULT E-cad(+) cell strains Ecad-231-7 and Ecad-231-9 were established. When cultured in ultra-low-binding dishes Ecad-231 cells grow in suspension while Ecad-231-7 and Ecad-231-9 cells grow in large clamps. When co-cultured with HCT116 cells, the average adhesion rates at 30 min are 39.0%, 60.0% and 59.5% for MDA-MB-231, Ecad-231-7 and Ecad-231-9 respectively. The average detachment rates by EDTA for 5 min are 37.4%, 4.2% and 7.4% respectively. So E-cad expression enhanced hemotypic and heterotypic cell-cell adhesion and cell-matrix adhesion. Forced exogenously expressed E-cad could combine with endogenous β-cat, whereas down stream cyclin D1 expression was significantly decreased, as evidenced by Western blot. The rates of cell apoptosis of MDA-MB-231, Ecad-231-7 and Ecad-231-9 were 1.8%, 2.0% and 2.1%. Expression of E-cad had no obvious effect on the apoptosis of tumor cells with regular culture. β-cat increased in the cytoplasma. CONCLUSIONS Two monoclonal tumor cell strains (Ecad-231-7 and Ecad-231-9) stably expressing E-cad were successfully established. E-cad could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involving β-cat and cyclin D1.