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

doi:10.3877/cma.j.issn.1674-0807.2014.01.004

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:N-cadherint=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。

关键词: 乳腺肿瘤, 细胞低氧, 上皮间质转化, beclin 1

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 including β-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-3β was decreased(F=36.096,P=0.000)under hypoxic condition for 24 h.

Conclusions

Under shortterm 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.

Key words: Breast neoplasms, Cell hypoxia, Epithelial-mesenchymal transition, beclin 1

图1 倒置荧光显微镜下观察慢病毒感染BT549 细胞效率(DAPI 染核 ×200) a,b:实验对照组;c,d:实验组

图2 Western blot 检测细胞beclin 1 蛋白表达情况 a:空白对照组;b:实验对照组;c:实验组

表1 各组细胞低氧培养12、24 h 后EMT 相关标志物相对mRNA 表达水平

组别	培养12 h后				培养24 h后
组别	E-cadherin	N-cadherin	vimentin	fibronectin	E-cadherin	N-cadherin	vimentin	fibronectin
空白对照组	1.067±0.111	1.074±0.070	1.078±0.070	1.080±0.075	0.767±0.050	1.215±0.038	1.416±0.059	1.526±0.053
实验对照组	1.079±0.037	1.087±0.067	0.933±0.049	1.181±0.091	0.792±0.030	1.213±0.039	1.393±0.039	1.609±0.045
实验组	1.828±0.018	0.458±0.056	0.339±0.054	0.270±0.041	1.190±0.035	0.817±0.057	0.749±0.035	0.630±0.040
F值	122.451	92.918	135.313	144.275	108.783	76.064	206.576	411.405
P值	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000

表1 各组细胞低氧培养12、24 h 后EMT 相关标志物相对mRNA 表达水平

组别	培养12 h后				培养24 h后
组别	E-cadherin	N-cadherin	vimentin	fibronectin	E-cadherin	N-cadherin	vimentin	fibronectin
空白对照组	1.067±0.111	1.074±0.070	1.078±0.070	1.080±0.075	0.767±0.050	1.215±0.038	1.416±0.059	1.526±0.053
实验对照组	1.079±0.037	1.087±0.067	0.933±0.049	1.181±0.091	0.792±0.030	1.213±0.039	1.393±0.039	1.609±0.045
实验组	1.828±0.018	0.458±0.056	0.339±0.054	0.270±0.041	1.190±0.035	0.817±0.057	0.749±0.035	0.630±0.040
F值	122.451	92.918	135.313	144.275	108.783	76.064	206.576	411.405
P值	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000

图3 各组细胞分别在低氧培养12 h 和24 h 后EMT 相关标志物mRNA 的表达变化 a:E-cadherin; b:N-cadherin; c:vimentin; d:fibronectin

表2 各组细胞低氧培养12、24 h 后EMT 相关标志物蛋白表达

组别	培养12 h后			培养24 h后
组别	E-cadherin	N-cadherin	vimentin	E-cadherin	N-cadherin	vimentin
空白对照组	0.326±0.023	0.687±0.021	0.421±0.039	0.183±0.011	0.839±0.030	0.616±0.023
实验对照组	0.288±0.040	0.723±0.009	0.426±0.032	0.198±0.018	0.800±0.023	0.610±0.043
实验组	0.494±0.040	0.552±0.003	0.231±0.017	0.306±0.030	0.604±0.045	0.410±0.015
F值	29.651	138.034	39.765	41.232	40.614	46.658
P值	0.001	0.000	0.000	0.000	0.000	0.000

表2 各组细胞低氧培养12、24 h 后EMT 相关标志物蛋白表达

组别	培养12 h后			培养24 h后
组别	E-cadherin	N-cadherin	vimentin	E-cadherin	N-cadherin	vimentin
空白对照组	0.326±0.023	0.687±0.021	0.421±0.039	0.183±0.011	0.839±0.030	0.616±0.023
实验对照组	0.288±0.040	0.723±0.009	0.426±0.032	0.198±0.018	0.800±0.023	0.610±0.043
实验组	0.494±0.040	0.552±0.003	0.231±0.017	0.306±0.030	0.604±0.045	0.410±0.015
F值	29.651	138.034	39.765	41.232	40.614	46.658
P值	0.001	0.000	0.000	0.000	0.000	0.000

表3 各组细胞低氧培养24 h 后EMT 相关标志物蛋白荧光强度

组别	E-cadherin	N-cadherin	vimentin
空白对照组	28 385.496±4143.292	28 275.775±5505.788	69 393.337±9212.869
实验对照组	28 804.766±7691.387	25 488.893±5167.678	68 975.898±10 373.393
实验组	40 738.018±5343.637	11 933.560±2269.632	46 768.475±2379.603
F值	5.629	14.753	10.146
P值	0.026	0.001	0.005

表3 各组细胞低氧培养24 h 后EMT 相关标志物蛋白荧光强度

组别	E-cadherin	N-cadherin	vimentin
空白对照组	28 385.496±4143.292	28 275.775±5505.788	69 393.337±9212.869
实验对照组	28 804.766±7691.387	25 488.893±5167.678	68 975.898±10 373.393
实验组	40 738.018±5343.637	11 933.560±2269.632	46 768.475±2379.603
F值	5.629	14.753	10.146
P值	0.026	0.001	0.005

图4 Western blot 检测各组细胞低氧培养12 h 和24 h 后EMT 相关标志物蛋白表达情况 a:空白对照组;b:实验对照组;c:实验组

图5 各组细胞分别在低氧培养12 h 和24 h 后EMT 相关标志物蛋白的表达变化 a:E-cadherin; b:N-cadherin; c:vimentin

图6 激光共聚焦显微镜观察各组细胞低氧培养24 h 后EMT 相关标志物蛋白表达( ×600) a:空白对照组;b:实验对照组;c:实验组AF594 为荧光二抗Alexa Fluor 594 标记目的蛋白,DAPI 染核,Merge 为合成图像

表4 各组细胞低氧培养24 h 后Wnt/β-catenin信号通路相关蛋白表达

组别	β-catenin	Snail	p-GSK-3β
空白对照组	0.553±0.038	0.298±0.056	0.244±0.029
实验对照组	0.554±0.032	0.302±0.036	0.219±0.030
实验组	0.369±0.055	0.168±0.018	0.390±0.018
F值	18.169	11.098	36.096
P值	0.003	0.010	0.000

表4 各组细胞低氧培养24 h 后Wnt/β-catenin信号通路相关蛋白表达

组别	β-catenin	Snail	p-GSK-3β
空白对照组	0.553±0.038	0.298±0.056	0.244±0.029
实验对照组	0.554±0.032	0.302±0.036	0.219±0.030
实验组	0.369±0.055	0.168±0.018	0.390±0.018
F值	18.169	11.098	36.096
P值	0.003	0.010	0.000

图7 Western blot 检测各组细胞低氧培养24 h 后Wnt/βcatenin 信号通路相关蛋白表达 a:空白对照组;b:实验对照组;c:实验组

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

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