【摘要】：目的:本研究旨在探討氟維司群(ICI 182780,ICI)對(duì)乳腺癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)表型的影響及其信號(hào)機(jī)制,為探尋治療乳腺癌的高效方法提供實(shí)驗(yàn)依據(jù)。方法:以雌激素受體(Estrogen receptor,ER)陽(yáng)性、G蛋白偶聯(lián)雌激素受體(G protein-coupled estrogen receptor,GPER)陽(yáng)性乳腺癌細(xì)胞MCF-7和ER陰性、GPER陽(yáng)性乳腺癌細(xì)胞MDA-MB-468為研究模型;常規(guī)細(xì)胞培養(yǎng),以ICI(E2作為對(duì)照)刺激處理模型細(xì)胞,需要時(shí)用GPER抑制劑G15預(yù)處理細(xì)胞;采用劃痕修復(fù)和侵襲小室實(shí)驗(yàn)分別考察細(xì)胞遷移和侵襲能力;通過(guò)蛋白印跡法檢測(cè)細(xì)胞蛋白表達(dá)水平;Sh-RNA-CANP2轉(zhuǎn)染細(xì)胞以沉默其基因的表達(dá),嘌呤霉素篩選穩(wěn)定表達(dá)細(xì)胞株。結(jié)果:(1)ICI(10μM)或E2(50nM)刺激模型細(xì)胞,可明顯增強(qiáng)MCF-7細(xì)胞遷移率(P0.05 vs DMSO)和侵襲率(P0.01 vs DMSO);MDA-MB-468細(xì)胞遷移率和侵襲率也明顯升高(P0.01 vs DMSO)。(2)ICI或E2處理模型細(xì)胞,MCF-7細(xì)胞纖連蛋白(Fibronectin,FN)表達(dá)明顯上調(diào)(P0.05,P0.01 vs DMSO),E-鈣黏素(E-cadherin,E-Cad)表達(dá)下調(diào)(P0.01,P0.05 vs DMSO);MDA-MB-468細(xì)胞FN表達(dá)也明顯上調(diào),E-Cad表達(dá)下調(diào)(P0.05 vs DMSO)。(3)給予G15(10μM)預(yù)處理模型細(xì)胞后,MCF-7細(xì)胞ICI或E2處理組遷移率均下降(P0.05,P0.01 vs ICI),侵襲率降低(P0.01,P0.05 vs ICI);MDA-MB-468細(xì)胞遷移率降低(P0.05,P0.01 vs ICI),侵襲率也降低(P0.05 vs ICI)。(4)G15還可明顯抑制ICI或E2誘導(dǎo)的模型細(xì)胞FN的上調(diào)(P0.01vs ICI或E2),MDA-MB-468細(xì)胞E-cad的下調(diào)(P0.05 vs ICI或E2),但對(duì)MCF-7細(xì)胞E-cad表達(dá)無(wú)明顯影響(P0.05 vs ICI或E2)。(5)Sh-RNA轉(zhuǎn)染沉默模型細(xì)胞CANP2表達(dá)可明顯降低細(xì)胞遷移和侵襲能力(P0.05 vs NCSH)。(6)CANP2沉默可下調(diào)模型細(xì)胞FN表達(dá)(P0.01)(P0.05 vs NCSH),上調(diào)細(xì)胞E-cad的表達(dá)(P0.01 vs NCSH)。(7)Sh-RNA轉(zhuǎn)染沉默模型細(xì)胞CANP2表達(dá),E2或ICI誘導(dǎo)的MCF-7細(xì)胞遷移率和侵襲率均下降(P0.05 vs NCSH);MDA-MB-468細(xì)胞兩處理組遷移率下降(P0.05 vs NCSH),侵襲率降低(P0.01 vs NCSH)。(8)CANP2表達(dá)沉默,E2或ICI誘導(dǎo)的模型細(xì)胞FN的上調(diào)及E-cad表達(dá)的下調(diào)被明顯抑制(P0.05 vs NCSH)。結(jié)論:ICI能誘導(dǎo)乳腺癌細(xì)胞從EMT野生型轉(zhuǎn)變?yōu)橥耆突虿煌耆?同時(shí)伴隨細(xì)胞遷移和侵襲能力明顯增強(qiáng);GPER-CANP2信號(hào)通路參與介導(dǎo)ICI誘導(dǎo)的EMT表型及細(xì)胞惡性生物學(xué)行為的變化。
[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.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.9

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