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-GTP-beclin 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 N-cadherin、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：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)；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：N-cadherin t=2．805、P=0．049、t=5．352、P=0．006,vimentin t=12．701、P=0．000、t=5．923、P=0．004,fibronectin t=7．318、P=0．002；实验组：N-cadherin t=7．849、P=0．001、t=1．987、P=0．184,vimentin t=11．097、P=0．000、t=13．626、P=0．000,fibronectin t=11．003、P=0．000)；beclin 1基因与低氧环境对 BT549细胞E-cadherin、N-cadherin的表达水平有交互效应( E-cadherin： F=19．175、P=0．000,F=5．588、P=0．015；N-cadherin：F=8．238、P=0．006,F=5．934、P=0．016)；低氧培养24 h后,实验组Wnt/β-catenin信号通路的β-catenin、Snail蛋白水平下调(β-catenin： F=18．169, P=0．003；Snail： F=11．098, P=0．010),而p-GSK-3β的蛋白水平上调(F=36．096, P=0．000)。结论 beclin 1基因在短程低氧条件下抑制BT549细胞EMT,而随时间的增加低氧促进 EMT；beclin 1基因在短程低氧环境下抑制 EMT 的机制可能与Wnt/β-catenin信号通路有关。
目的： beclin 1基因慢病毒錶達載體穩定感染三陰性乳腺癌BT549細胞後,于短程低氧條件下探討beclin 1基因對上皮間質轉化( EMT)的影響。方法用帶有pLenO-GTP Vector及pLenO-GTP-beclin 1 Vector的慢病毒以感染複數( MOI)為20分彆感染BT549細胞,即實驗對照組及實驗組,未感染細胞作為空白對照。感染96 h後用倒置熒光顯微鏡觀察熒光,估計感染效率；用Western blot法檢測beclin 1基因是否在 BT549細胞中穩定錶達；將各組細胞低氧培養12、24 h 後採用熒光定量 PCR、Western blot法及激光共聚焦顯微鏡檢測EMT相關標誌物；用Western blot法檢測低氧培養24 h後的各組細胞Wnt/β-catenin信號通路的相關蛋白錶達水平。用兩樣本t檢驗分析低氧時間對細胞EMT相關標誌物的錶達情況,餘計量資料用方差分析進行統計。結果慢病毒感染BT549細胞96 h後,感染效率約為100%,實驗組beclin 1蛋白高效穩定錶達(F=107．200,P=0．000)；低氧條件下,相對于空白對照組、實驗對照組,實驗組細胞的上皮標誌物E-cadherin mRNA和蛋白錶達水平上調(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),而間皮標誌物 N-cadherin、vimentin及 fibronectin錶達水平下調(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)；相對于低氧培養12 h,各組細胞低氧培養24 h後E-cadherin錶達水平下調(空白對照組：t=4．266、P=0．013,t=11．235、P=0．000；實驗對照組：t=10．463、P=0．000,t=4．092、P=0．009；實驗組：t=28．208、P=0．000,t=7．262、P=0．001),而 N-cadherin(實驗組除外)、vimentin和fibronectin錶達水平上調(空白對照組：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；實驗對照組：N-cadherin t=2．805、P=0．049、t=5．352、P=0．006,vimentin t=12．701、P=0．000、t=5．923、P=0．004,fibronectin t=7．318、P=0．002；實驗組：N-cadherin t=7．849、P=0．001、t=1．987、P=0．184,vimentin t=11．097、P=0．000、t=13．626、P=0．000,fibronectin t=11．003、P=0．000)；beclin 1基因與低氧環境對 BT549細胞E-cadherin、N-cadherin的錶達水平有交互效應( E-cadherin： F=19．175、P=0．000,F=5．588、P=0．015；N-cadherin：F=8．238、P=0．006,F=5．934、P=0．016)；低氧培養24 h後,實驗組Wnt/β-catenin信號通路的β-catenin、Snail蛋白水平下調(β-catenin： F=18．169, P=0．003；Snail： F=11．098, P=0．010),而p-GSK-3β的蛋白水平上調(F=36．096, P=0．000)。結論 beclin 1基因在短程低氧條件下抑製BT549細胞EMT,而隨時間的增加低氧促進 EMT；beclin 1基因在短程低氧環境下抑製 EMT 的機製可能與Wnt/β-catenin信號通路有關。
목적： beclin 1기인만병독표체재체은정감염삼음성유선암BT549세포후,우단정저양조건하탐토beclin 1기인대상피간질전화( EMT)적영향。방법용대유pLenO-GTP Vector급pLenO-GTP-beclin 1 Vector적만병독이감염복수( MOI)위20분별감염BT549세포,즉실험대조조급실험조,미감염세포작위공백대조。감염96 h후용도치형광현미경관찰형광,고계감염효솔；용Western blot법검측beclin 1기인시부재 BT549세포중은정표체；장각조세포저양배양12、24 h 후채용형광정량 PCR、Western blot법급격광공취초현미경검측EMT상관표지물；용Western blot법검측저양배양24 h후적각조세포Wnt/β-catenin신호통로적상관단백표체수평。용량양본t검험분석저양시간대세포EMT상관표지물적표체정황,여계량자료용방차분석진행통계。결과만병독감염BT549세포96 h후,감염효솔약위100%,실험조beclin 1단백고효은정표체(F=107．200,P=0．000)；저양조건하,상대우공백대조조、실험대조조,실험조세포적상피표지물E-cadherin mRNA화단백표체수평상조(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),이간피표지물 N-cadherin、vimentin급 fibronectin표체수평하조(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)；상대우저양배양12 h,각조세포저양배양24 h후E-cadherin표체수평하조(공백대조조：t=4．266、P=0．013,t=11．235、P=0．000；실험대조조：t=10．463、P=0．000,t=4．092、P=0．009；실험조：t=28．208、P=0．000,t=7．262、P=0．001),이 N-cadherin(실험조제외)、vimentin화fibronectin표체수평상조(공백대조조：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；실험대조조：N-cadherin t=2．805、P=0．049、t=5．352、P=0．006,vimentin t=12．701、P=0．000、t=5．923、P=0．004,fibronectin t=7．318、P=0．002；실험조：N-cadherin t=7．849、P=0．001、t=1．987、P=0．184,vimentin t=11．097、P=0．000、t=13．626、P=0．000,fibronectin t=11．003、P=0．000)；beclin 1기인여저양배경대 BT549세포E-cadherin、N-cadherin적표체수평유교호효응( E-cadherin： F=19．175、P=0．000,F=5．588、P=0．015；N-cadherin：F=8．238、P=0．006,F=5．934、P=0．016)；저양배양24 h후,실험조Wnt/β-catenin신호통로적β-catenin、Snail단백수평하조(β-catenin： F=18．169, P=0．003；Snail： F=11．098, P=0．010),이p-GSK-3β적단백수평상조(F=36．096, P=0．000)。결론 beclin 1기인재단정저양조건하억제BT549세포EMT,이수시간적증가저양촉진 EMT；beclin 1기인재단정저양배경하억제 EMT 적궤제가능여Wnt/β-catenin신호통로유관。
Objective 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. 013; t=11. 235, P=0. 000;negative control:t=10. 463, P=0. 000; t=4. 092, P=0. 009;experimental group:t=28. 208, P=0. 000; t=7. 262, P=0. 001)and the levels of N-cadherin(except the experimental group), vimentin and fibronectin were upregulated(blank control: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;negative control:N-cadherin t=2. 805, P=0. 049;t=5. 352, P=0. 006; vimentin t=12. 701, P=0. 000; t=5. 923, P=0. 004; fibronectin t=7. 318, P=0. 002;experimental group:N-cadherin t=7. 849, P=0. 001; t=1. 987, P=0. 184; vimentin t=11. 097, P=0. 000;t=13. 626, P=0. 000;fibronectin t=11. 003, P=0. 000) in these groups under hypoxic condition for 24 h. For the expression of E-cadherin and N-cadherin, there was an interaction between beclin 1 and hypoxic stimulation (E-cadherin:F=19. 175, P=0. 000;F=5. 588, P=0. 015;N-cadherin: F=8. 238, P=0. 006;F=5. 934, P=0. 016). Expressions of Wnt/β-catenin related proteins includingβ-catenin, snail were increased in experimental group(β-catenin: F=18. 169, P=0. 003; Snail: F=11. 098, P=0. 010), while p-GSK-3βwas decreased(F=36. 096, P=0. 000)under hypoxic condition for 24 h. Conclusions Under short-term hypoxic condition, beclin 1 gene inhibits EMT of BT-549 cells, but hypoxia promotes EMT with the increased hypoxic time. Wnt/β-catenin signaling pathway may be involved in the mechanism of EMT inhibition of beclin 1 gene under hypoxic condition.
