摘要
目的 :探究转谷氨酰胺酶1(transglutaminase 1,TGM1)基因过表达对乳腺癌细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)的影响。方法 :采用实时荧光定量PCR法检测鼠源性乳腺癌FE1.2和FE1.3细胞中TGM1 m RNA的表达水平。采用脂质体法将携带有TGM1基因全长序列的重组载体质粒p CMV3-TGM1-GFPspark转染至FE1.2细胞中,构建TGM1基因过表达的FE1.2细胞株(FE1.2-TGM1)。光学显微镜下观察FE1.2细胞形态的变化,FCM法测量细胞粒径的大小;MTT法检测TGM1基因过表达对鼠乳腺癌细胞增殖能力的影响;实时荧光定量PCR法和蛋白质印迹法分别检测EMT相关分子标志物上皮型钙黏蛋白(E-cadherin)、波形蛋白(vimentin)、β-链蛋白(β-catenin)以及细胞周期调节蛋白D1(cyclin D1)m RNA和蛋白的表达水平。最后,采用细胞划痕愈合实验检测TGM1过表达对细胞迁移能力的影响。结果 :FE1.3细胞中TGM1 m RNA的表达水平明显高于FE1.2细胞(P<0.001)。将重组载体p CMV3-TGM1-GFPspark转入FE1.2细胞后,FE1.2细胞中TGM1 m RNA和蛋白的表达水平明显上调(P<0.001和P<0.01),细胞形态发生明显改变,细胞的粒径增大(P<0.01),而FE1.2细胞的增殖能力明显降低(P<0.001)。TGM1基因过表达的E1.2细胞中E-cadherin m RNA的表达水平明显上调(P<0.01),vimentin m RNA及蛋白的表达水平均明显下调(P值均<0.001),cyclin D1 m RNA的表达水平明显上调(P<0.05),而cyclin D1蛋白表达水平无明显变化(P>0.05),β-catenin m RNA的表达水平无明显变化(P>0.05),β-catenin蛋白的表达水平明显下调(P<0.01);TGM1过表达后FE1.2细胞的迁移能力明显减弱(P<0.05)。结论 :TGM1基因过表达能抑制乳腺癌细胞的EMT。
Objective： To investigate the effect of transglutaminase 1（TGM 1） gene overexpression on the epithelial-mesenchymal transition（EMT） in breast cancer cells.Methods： The expression level of TGM1 m RNA in rat breast cancer FE1.2 and FE1.3 cells was detected by real-time fluorescent quantitative PCR. The recombinant vector p CMV3-TGM1-GFPspark was transfected into FE1.2 cells by liposome method to construct the TGM1 gene overexpressed FE1.2 cell strain. The morphological change of FE1.2 cells after transfection with p CMV3-TGM1-GFPspark was observed under an optical microscope, the cell size was detected by FCM method. The effect of TGM 1 gene overexpression on the proliferation of FE1.2 cells was examined by MTT method. The expression levels of EMT molecular markers E-cadherin, vimentin, β-catenin, cyclin D1 m RNAs and proteins were examined by real-time fluorescent quantitative PCR and Western blotting, respectively. Finally, the migration ability of FE1.2 cells transfected with p CMV3-TGM1-GFPspark was detected by wound healing assay.Results： The expression level of TGM1 m RNA in FE1.3 cells was significantly higher than that in FE1.2 cells（P〈0.001）. After the recombinant vector p CMV3-TGM1-GFPspark was transfected into FE1.2 cells, the expression levels of TGM1 m RNA and protein were significantly up-regulated（both P〈0.001）; the shape of FE1.2 cells was changed, and the diameter of FE1.2 cells was enlarged（P〈0.01）. As compared with the FE1.2 cells（blank control） and FE1.2-vector group, the proliferation ability of FE1.2 cells transfected with p CMV3-TGM1-GFPspark was significantly inhibited（both P〈0.001）, the m RNA level of E-cadherin was up-regulated（P〈0.01）, the expression levels of vimentin m RNA and protein and β-catenin protein were down-regulated（all P〈0.01）, the expression level of cyclin D1 m RNA was up-regulated（P〈0.05）, the expression levels of cyclin D1 protein and β-catenin m RNA were not significantly changed（both P〈0.05）. The migration ability of FE1.2 cells after TGM1 gene overexpression was significantly inhibited（P〈0.05）.Conclusion： Overexpression of TGM 1 gene can inhibit EMT in breast cancer cells.
作者
宋喻
姚尧
夏雷
肖潇
刘务玲
杨珏
刘唐婧君
宋晶睿
宋佳蕾
王宁
刘杰麟
YAACOV Ben-David
SONG Yu;YAO Yao;XlA Lei;XlAO Xiao;LIU Wuling;YANG Jue;LIU-TANG Jingjun;SONG Jingrui;SONG Jialei;WANG Ning;LIU Jielin;YAACOV Ben-David(Department of Immunology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou Province,China;Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences,Guiyang 550014,Guizhou Province,China;State Key Laboratory for Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550025,Guizhou Province,China;Department of Pharmacology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou Province,China;College of Bioengineering and Faculty of Sciences,Chongqing University,Chongqing 400044,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2018年第7期670-679,共10页
Tumor
基金
国家自然科学基金资助项目(编号：81472609)
贵州省科技合作计划资助项目[编号：黔科合LH字(2015)7403]
