短程低氧条件下乳腺癌BT549细胞表达beclin 1基因对上皮间质转化的影响  

Influence on epithelial-mesenchymal transition by transfecting beclin 1 in breast cancer BT-549 cells under short-term hypoxic condition

作　　者：郑临海[1] 吴爱国 范旭龙[1] 王梦川[1] 邵国利[1] 纪术峰[1] 焦庆丽 

机构地区：[1]南方医科大学珠江医院普外科,广州510282

出　　处：《中华乳腺病杂志（电子版）》2014年第1期13-18,共6页Chinese Journal of Breast Disease(Electronic Edition)

基　　金：广东省自然科学基金资助项目(S2012010009276)

摘　　要：目的 beclin 1基因慢病毒表达载体稳定感染三阴性乳腺癌BT549细胞后,于短程低氧条件下探讨beclin 1基因对上皮间质转化(EMT)的影响。方法用带有pLenO-GTP Vector及pLenO-GTPbeclin 1 Vector的慢病毒以感染复数(MOI)为20分别感染BT549细胞,即实验对照组及实验组,未感染细胞作为空白对照。感染96 h后用倒置荧光显微镜观察荧光,估计感染效率;用Western blot法检测beclin 1基因是否在BT549细胞中稳定表达;将各组细胞低氧培养12、24 h后采用荧光定量PCR、Western blot法及激光共聚焦显微镜检测EMT相关标志物;用Western blot法检测低氧培养24 h后的各组细胞Wnt/β-catenin信号通路的相关蛋白表达水平。用两样本t检验分析低氧时间对细胞EMT相关标志物的表达情况,余计量资料用方差分析进行统计。结果慢病毒感染BT549细胞96 h后,感染效率约为100%,实验组beclin 1蛋白高效稳定表达(F=107.200,P=0.000);低氧条件下,相对于空白对照组、实验对照组,实验组细胞的上皮标志物E-cadherin mRNA和蛋白表达水平上调(12 h:F=122.451、P=0.000,F=29.651、P=0.001;24 h:F=108.783、P=0.000,F=41.232、P=0.000),而间皮标志物Ncadherin、vimentin及fibronectin表达水平下调(12 h:N-cadherin F=92.918、P=0.000、F=138.034、P=0.000,vimentin F=135.313、P=0.000、F=39.765、P=0.000,fibronectin F=144.275、P=0.000;24 h:Ncadherin F=76.064、P=0.000、F=40.614、P=0.000,vimentin F=206.576、P=0.000、F=46.658、P=0.000,fibronectin F=411.405、P=0.000);相对于低氧培养12 h,各组细胞低氧培养24 h后E-cadherin表达水平下调(空白对照组:t=4.266、P=0.013,t=11.235、P=0.000;实验对照组:t=10.463、P=0.000,t=4.092、P=0.009;实验组:t=28.208、P=0.000,t=7.262、P=0.001),而N-cadherin(实验组除外)、vimentin和fibronectin表达水平上调(空白对照组:N-cadherin t=3.072、P=0.037、t=7.146、P=0.002,vimentin t=6.384、P=0.003、t=7.476、P=0.002,fibronectin t=8.389、P=0.001;实验对照组:Ncadherin t=2Objective To study the effect of beclin 1 gene on epithelial-mesenchymal transition （EMT） in triple-negative breast cancer BT-549 cells under short-term hypoxic condition. Methods The pLenO-GTP-beclin 1 and pLenO-GTP lentivirus were infected into triple-negative breast cancer BT-549 cells respectively with （ multiplicity of infection, MOI ）= 20, which served as experimental group and negative control, uninfected BT-549 cells as blank control. The efficiency was evaluated by fluorescence at 96 h after infection, and beclin 1 protein were detected by Western blot. Then the cells were cultured under hypoxic condition for 12 h and 24 h. The mRNA levels of EMT markers were detected by RT-PCR, protein levels were detected by Western blot and laser scanning confocal microscope. At last, the levels of Wnt/β-catenin related proteins were detected by Western blot in three groups under hypoxic condition for 24 h. The effect of hypoxic time on EMT was analyzed by two-sample t-test, the other measurement data were analyzed using analysis of variance （ ANOVA ） . Results The efficiency was approximately 100% at 96 h after infection, and the expression of beclin 1 protein was increased evidently in experimental group（F=107.200, P=0.000）. Compared with negative control and blank control, the mRNA and protein levels of E-cadherin （ epithelial marker） were upregulated in experimental group under hypoxic condition（12 h：F=122. 451, P=0. 000; F=29. 651, P=0. 001;24 h：F=108. 783, P=0. 000; F=41. 232, P=0. 000）, but the levels of N-cadherin, vimentin and fibronectin（mesenchymal markers） were downregulated（12 h：N-cadherin F=92. 918, P=0. 000;F=138. 034, P=0. 000;vimentin F=135. 313, P=0. 000;F=39. 765, P=0. 000;fibronectin F=144. 275, P=0. 000;24 h：N-cadherin F=76. 064, P=0. 000; F=40. 614, P=0. 000; vimentin F=206. 576, P=0. 000;F=46. 658, P=0. 000; fibronectin F=411. 405, P=0. 000）. Compared with 12 h, the mRNA and protein levels of E-cadherin were downregulated （blank control：t=4. 266, P=0.

关 键 词：乳腺肿瘤 细胞低氧 上皮间质转化 BECLIN1 

分 类 号：R737.9[医药卫生—肿瘤]