远华蟾毒精对体外乳腺癌4T1细胞上皮间质转化的影响  

The effect and mechanism of telocinobufagin on the epithelial mesenchymal transition of breast cancer 4T1 cells in vitro

作　　者：高玉雪 曹珍 吕世军[1] 朱学涛 李峰[1] 王茹燕 徐金媛 钟瑾怡 史立宏[1] GAO Yuxue;CAO Zhen;LV Shijun;ZHU Xuetao;LI Feng;WANG Ruyan;XU Jinyuan;ZHONG Jinyi;SHI Lihong(Wei fang Medical University, Wei fang 261053, Shandong, China)

机构地区：[1]潍坊医学院,山东潍坊261053

出　　处：《西部医学》2018年第6期809-812,818,共5页Medical Journal of West China

基　　金：山东省自然科学基金(ZR2014HM086)

摘　　要：目的观察远华蟾毒精对乳腺癌细胞4T1迁移、侵袭及上皮-间质转化(epithelial-mesenchymal transition,EMT)的影响,并研究其相关机制。方法将鼠乳腺癌细胞系4T1细胞与不同浓度远华蟾毒精(0、0.05、0.1、0.5、1、1.25和1.5μg/ml)分别培养24h后,MTT试验检测远华蟾毒精对4T1增殖活性的影响;将实验分为对照组(0μg/ml)和实验组(0.05、0.5μg/ml),细胞划痕实验和Transwell实验检测对照组(0μg/ml)和实验组(0.05、0.5μg/ml)对细胞迁移、侵袭能力的影响;免疫荧光检测对照组(0μg/ml)和实验组(0.5μg/ml)上皮间质转化相关蛋白E-钙粘蛋白(E-cadherin)、波形蛋白(Vimentin)、纤维链接蛋百(Fibronectin)相关蛋白的变化;蛋白质印迹法检测对照组(0μg/ml)和实验组(0.05、0.5μg/ml)上皮间质转化相关蛋白E-cadherin、Vimentin、Fibronectin、转录因子Snail以及PI3k/Akt/mTOR信号通路相关蛋白p-Akt、p-mTOR表达的变化。结果MTT试验结果显示,低浓度远华蟾毒精(0、0.05、0.1、0.5和1μg/ml)对4T1增殖无抑制作用,高浓度远华蟾毒精(1.25和1.5μg/ml)对4T1增殖有抑制作用;细胞划痕实验结果显示,对照组与实验组细胞迁移率差异均有统计学意义(P<0.05);Transwell迁移实验结果显示,对照组与实验组穿至下室的细胞数差异均有统计学意义(P<0.05);Transwell侵袭实验结果显示,对照组与实验组穿至下室的细胞数差异均有统计学意义(P<0.05);免疫荧光结果显示,实验组与对照组相比,E-cadherin表达增强,Vimentin和Fibronectin表达减弱(P<0.05);蛋白质印迹法结果显示,E-cadherin表达上调,Vimentin和Fibronectin表达下调(P<0.05);转录因子Snail表达下调(P<0.05);PI3k/Akt/mTOR信号通路相关蛋白p-Akt、p-mTOR表达下调(P<0.05)。结论远华蟾毒精通过Akt/mTOR/Snail信号通路抑制乳腺癌的迁移、侵袭及上皮-间质转化,为远华蟾毒精的临床应用提供了理论依据。Objective To observe the effect of Teloeinobufagin on migration, invasion and epithelial-mesenehyrnal transition （EMT） and mechanism research of breast cancer cell line 4T1. Methods 4T1 cells with different concentrations of Tcloeinobufagin （0, 0.05, 0.1, 0.5, 1, 1.25 and 1.5 μg/mL） were cultured for 24 h. MTT test was used to access to proliferation of 4T1 ceils. The experiment was divided into the control group （0 mu g/ml） and the experimental group （0.05, 0. 5 mu g/ml）. The migration and invasion ability was tested by wound healing assay and transwell assay. The expression levels of E-cadherin, Vimentin and Fibroneetin by immunofluorescence and western blot. The expression levels of Snail, p-Akt and p-roTOR by western blot. Results The MTT test showed that low concentration of Teloeinobufagin （0, 0. 05, 0.1, 0.5 and 1μg/mL） had no inhibitory effect on the proliferation of 4T1 cells, high concentration of Telocinobufagin （1.25 and 1.5μg/mL） had inhibitory effect on the proliferation of 4T1 ceils, wound healing assay revealed that cell migration rations were respectively （85.8 ±5.2） %, （49.3±4.5）% and （30.3±4.5）% （F= 105. 686, P〈0.05）. Transwell migration assay found that the cell number was respectivelyi81. 7±16. 7, 88± 11.6 and 32.7± 5.2 （F= 157. 909, P〈0.05）. Transwell invasion assay found that the cell number was respectively 142.2 ± 14. 9, 55.2 ± 9.3 and 27.5± 7.1 （ F = 118.766, P〈 0.05 ）. Immunofluorescence showed that E-cadherin was upregulated, Vimentin and Fibronectin were downregulated. Western blotting showed that the expression of E-cadherin was increased, P〈0.05; the expression of Vimentin, Fibronectin, transcription factor Snail, p-Akt, p-mTOR were decreased, P〈0. 05. Conclusion Telocinobufagin inhibited epithelial mesenchymal transition of breast cancer 4T1 cells via Akt/ mTOR/Snail pathway.

关 键 词：远华蟾毒精 乳腺癌 上皮间质转化 4T1细胞 

分 类 号：R737.9[医药卫生—肿瘤]