氟维司群对乳腺癌细胞EMT表型的影响及其信号机制研究
发布时间：2018-09-17 16:14
【摘要】：目的:本研究旨在探讨氟维司群(ICI 182780,ICI)对乳腺癌细胞上皮-间质转化(Epithelial-mesenchymal transition,EMT)表型的影响及其信号机制,为探寻治疗乳腺癌的高效方法提供实验依据。方法:以雌激素受体(Estrogen receptor,ER)阳性、G蛋白偶联雌激素受体(G protein-coupled estrogen receptor,GPER)阳性乳腺癌细胞MCF-7和ER阴性、GPER阳性乳腺癌细胞MDA-MB-468为研究模型;常规细胞培养,以ICI(E2作为对照)刺激处理模型细胞,需要时用GPER抑制剂G15预处理细胞;采用划痕修复和侵袭小室实验分别考察细胞迁移和侵袭能力;通过蛋白印迹法检测细胞蛋白表达水平;Sh-RNA-CANP2转染细胞以沉默其基因的表达,嘌呤霉素筛选稳定表达细胞株。结果:(1)ICI(10μM)或E2(50nM)刺激模型细胞,可明显增强MCF-7细胞迁移率(P0.05 vs DMSO)和侵袭率(P0.01 vs DMSO);MDA-MB-468细胞迁移率和侵袭率也明显升高(P0.01 vs DMSO)。(2)ICI或E2处理模型细胞,MCF-7细胞纤连蛋白(Fibronectin,FN)表达明显上调(P0.05,P0.01 vs DMSO),E-钙黏素(E-cadherin,E-Cad)表达下调(P0.01,P0.05 vs DMSO);MDA-MB-468细胞FN表达也明显上调,E-Cad表达下调(P0.05 vs DMSO)。(3)给予G15(10μM)预处理模型细胞后,MCF-7细胞ICI或E2处理组迁移率均下降(P0.05,P0.01 vs ICI),侵袭率降低(P0.01,P0.05 vs ICI);MDA-MB-468细胞迁移率降低(P0.05,P0.01 vs ICI),侵袭率也降低(P0.05 vs ICI)。(4)G15还可明显抑制ICI或E2诱导的模型细胞FN的上调(P0.01vs ICI或E2),MDA-MB-468细胞E-cad的下调(P0.05 vs ICI或E2),但对MCF-7细胞E-cad表达无明显影响(P0.05 vs ICI或E2)。(5)Sh-RNA转染沉默模型细胞CANP2表达可明显降低细胞迁移和侵袭能力(P0.05 vs NCSH)。(6)CANP2沉默可下调模型细胞FN表达(P0.01)(P0.05 vs NCSH),上调细胞E-cad的表达(P0.01 vs NCSH)。(7)Sh-RNA转染沉默模型细胞CANP2表达,E2或ICI诱导的MCF-7细胞迁移率和侵袭率均下降(P0.05 vs NCSH);MDA-MB-468细胞两处理组迁移率下降(P0.05 vs NCSH),侵袭率降低(P0.01 vs NCSH)。(8)CANP2表达沉默,E2或ICI诱导的模型细胞FN的上调及E-cad表达的下调被明显抑制(P0.05 vs NCSH)。结论:ICI能诱导乳腺癌细胞从EMT野生型转变为完全型或不完全型,同时伴随细胞迁移和侵袭能力明显增强;GPER-CANP2信号通路参与介导ICI诱导的EMT表型及细胞恶性生物学行为的变化。
[Abstract]:Objective: to investigate the effect of ICI 182780 on the epithelial-interstitial transformation (Epithelial-mesenchymal transition,EMT) phenotype of breast cancer cells and its signal mechanism, and to provide experimental evidence for the effective treatment of breast cancer. Methods: the model of Estrogen receptor,ER positive, G protein-coupled estrogen receptor (G protein-coupled estrogen receptor,GPER) positive breast cancer cell line MCF-7 and ER negative GPER positive breast cancer cell line MDA-MB-468 was used as the model, and the normal cell culture was treated with ICI (E2 as control). When necessary, the cells were pretreated with GPER inhibitor G15, the cell migration and invasion ability were investigated by scratch repair and invasive chamber experiments, and the cell protein expression level was detected by Western blotting to silence the expression of its gene in the cells transfected with Sh-RNA-CANP2. Purine mycin was used to screen stable expression cell lines. Results: (1) ICI (10 渭 M) or E2 (50nM) stimulated model cells, The migration rate and invasion rate of MCF-7 cells (P0.05 vs DMSO) and P0.01 vs DMSO) mDA-MB-468) were also significantly increased (P0.01 vs DMSO). (2). The expression of fibronectin (Fibronectin,FN) in MDA-MB-468 cells treated with ICI or E2 was significantly up-regulated (P0.05P0.01 vs DMSO) P0.01 vs DMSO) E-cadherin E-Cad). The FN expression of MDA-MB-468 cells was also significantly up-regulated (P0.05 vs DMSO). (3). After G15 (10 渭 M) pretreated with G15 (10 渭 M), the migration rates of MDA-MB-468 cells were significantly decreased (P0.05and P0.01 vs ICI), invasion rate (P0.01P0.05) vs ICI) MDA-MB-468 cell migration rate decreased (P0.05 P0.01 vs ICI), invasion) after G15 (10 渭 M) pretreatment of the model cell line MDA-MB-468 cells were treated with G15 (10 渭 M). The attack rate was also decreased (P0.05 vs ICI). (4) G15 significantly inhibited the up-regulation of FN (P0.01vs ICI or E2) induced by ICI or E2 in MDA-MB-468 cells (P0.05 vs ICI or E2), but had no effect on E-cad expression of MCF-7 cells (P0.05 vs ICI or E2). (5) Sh-RNA transfected the model cells CANP2. Expression of P05 vs NCSH). (6 CANP2 silencing could down-regulate the expression of FN (P0.01) (P0.05 vs NCSH), up-regulated the expression of E-cad (P0.01 vs NCSH). (7) Sh-RNA transfected with Sh-RNA induced CANP2 expression of E2 or ICI induced migration and invasion of MCF-7 cells. The rate of attack decreased (P0.05) the migration rate of MDA-MB-468 cells decreased (P0.05) the invasion rate of vs NCSH), decreased (P0.01 vs NCSH). (8) CANP2 expression silenced E2 or ICI induced FN and the down-regulation of E-cad expression was significantly inhibited (P0.05 vs NCSH). ConclusionICI can induce breast cancer cells to change from EMT wild type to complete type or incomplete type, and the ability of cell migration and invasion can be significantly enhanced by GPER-CANP2 signaling pathway involved in ICI induced changes in EMT phenotype and malignant biological behavior of breast cancer cells.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
本文编号：2246452
[Abstract]:Objective: to investigate the effect of ICI 182780 on the epithelial-interstitial transformation (Epithelial-mesenchymal transition,EMT) phenotype of breast cancer cells and its signal mechanism, and to provide experimental evidence for the effective treatment of breast cancer. Methods: the model of Estrogen receptor,ER positive, G protein-coupled estrogen receptor (G protein-coupled estrogen receptor,GPER) positive breast cancer cell line MCF-7 and ER negative GPER positive breast cancer cell line MDA-MB-468 was used as the model, and the normal cell culture was treated with ICI (E2 as control). When necessary, the cells were pretreated with GPER inhibitor G15, the cell migration and invasion ability were investigated by scratch repair and invasive chamber experiments, and the cell protein expression level was detected by Western blotting to silence the expression of its gene in the cells transfected with Sh-RNA-CANP2. Purine mycin was used to screen stable expression cell lines. Results: (1) ICI (10 渭 M) or E2 (50nM) stimulated model cells, The migration rate and invasion rate of MCF-7 cells (P0.05 vs DMSO) and P0.01 vs DMSO) mDA-MB-468) were also significantly increased (P0.01 vs DMSO). (2). The expression of fibronectin (Fibronectin,FN) in MDA-MB-468 cells treated with ICI or E2 was significantly up-regulated (P0.05P0.01 vs DMSO) P0.01 vs DMSO) E-cadherin E-Cad). The FN expression of MDA-MB-468 cells was also significantly up-regulated (P0.05 vs DMSO). (3). After G15 (10 渭 M) pretreated with G15 (10 渭 M), the migration rates of MDA-MB-468 cells were significantly decreased (P0.05and P0.01 vs ICI), invasion rate (P0.01P0.05) vs ICI) MDA-MB-468 cell migration rate decreased (P0.05 P0.01 vs ICI), invasion) after G15 (10 渭 M) pretreatment of the model cell line MDA-MB-468 cells were treated with G15 (10 渭 M). The attack rate was also decreased (P0.05 vs ICI). (4) G15 significantly inhibited the up-regulation of FN (P0.01vs ICI or E2) induced by ICI or E2 in MDA-MB-468 cells (P0.05 vs ICI or E2), but had no effect on E-cad expression of MCF-7 cells (P0.05 vs ICI or E2). (5) Sh-RNA transfected the model cells CANP2. Expression of P05 vs NCSH). (6 CANP2 silencing could down-regulate the expression of FN (P0.01) (P0.05 vs NCSH), up-regulated the expression of E-cad (P0.01 vs NCSH). (7) Sh-RNA transfected with Sh-RNA induced CANP2 expression of E2 or ICI induced migration and invasion of MCF-7 cells. The rate of attack decreased (P0.05) the migration rate of MDA-MB-468 cells decreased (P0.05) the invasion rate of vs NCSH), decreased (P0.01 vs NCSH). (8) CANP2 expression silenced E2 or ICI induced FN and the down-regulation of E-cad expression was significantly inhibited (P0.05 vs NCSH). ConclusionICI can induce breast cancer cells to change from EMT wild type to complete type or incomplete type, and the ability of cell migration and invasion can be significantly enhanced by GPER-CANP2 signaling pathway involved in ICI induced changes in EMT phenotype and malignant biological behavior of breast cancer cells.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
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