譚擁軍，陳含笑

(湖南大學(xué) 生物學(xué)院,湖南 長(zhǎng)沙 410082)

轉(zhuǎn)錄因子FOXM1剪接異構(gòu)體在乳腺癌EMT過(guò)程中的初步研究*

譚擁軍?，陳含笑

(湖南大學(xué) 生物學(xué)院,湖南 長(zhǎng)沙 410082)

探究轉(zhuǎn)錄因子FOXM1不同的剪接異構(gòu)體對(duì)乳腺癌EMT過(guò)程中的影響.采用基因工程方法分別構(gòu)建了表達(dá)FOXM1B-EGFP和FOXM1C-EGFP兩種 FOXM1剪接異構(gòu)體真核表達(dá)質(zhì)粒，并將其轉(zhuǎn)染進(jìn)乳腺癌細(xì)胞，采用RT-PCR和Western印跡檢測(cè)細(xì)胞樣本中FOXM1剪接異構(gòu)體的表達(dá)和EMT相關(guān)基因表達(dá)，同時(shí)采用transwell檢測(cè)高表達(dá)不同F(xiàn)OXM1剪接異構(gòu)體細(xì)胞的侵襲和遷移能力.成功構(gòu)建了FOXM1B-EGFP和FOXM1C-EGFP真核表達(dá)質(zhì)粒.外源FOXM1B在乳腺癌間質(zhì)型細(xì)胞的表達(dá)高于上皮型細(xì)胞，并主要存在于細(xì)胞核內(nèi)，且高表達(dá)FOXM1B能夠顯著促進(jìn)乳腺癌細(xì)胞的侵襲和EMT過(guò)程.外源FOXM1C在乳腺癌上皮型細(xì)胞中的表達(dá)高于間質(zhì)型細(xì)胞，并且在細(xì)胞核和細(xì)胞質(zhì)中均有表達(dá)，高表達(dá)FOXM1C能夠顯著抑制細(xì)胞的侵襲和EMT過(guò)程.實(shí)驗(yàn)結(jié)果表明，在乳腺癌細(xì)胞中，F(xiàn)OXM1B主要存在于細(xì)胞核內(nèi)，F(xiàn)OXM1C在細(xì)胞核和細(xì)胞質(zhì)中均有表達(dá).本研究預(yù)示FOXM1B和FOXM1C對(duì)乳腺癌細(xì)胞EMT過(guò)程發(fā)揮不同的影響.

細(xì)胞粘附；FOXM1B；FOXM1C；乳腺癌； EMT

FOXM1(Forkhead box protein M1)是叉頭框(Forkhead box，F(xiàn)ox)轉(zhuǎn)錄因子家族中的成員，又稱(chēng)為T(mén)rident,HFH-11,Win,MPP-2等，定位于 12p13-3 號(hào)染色體，由十個(gè)外顯子組成[1].FOXM1的蛋白表達(dá)存在著A,B 和C 3種剪接異構(gòu)體：FOXM1A 比FOXM1B多兩個(gè)外顯子，不具有轉(zhuǎn)錄活性，在細(xì)胞中表達(dá)很低；FOXM1C比FOXM1B多一個(gè)外顯子，和FOXM1B一樣具有轉(zhuǎn)錄活性，且在腫瘤組織中有高表達(dá)，但在某些方面具有不同的生物學(xué)功能[2-3]；FOXM1B主要在癌細(xì)胞中高表達(dá)，而FOXM1C在正常細(xì)胞和癌細(xì)胞中均有高表達(dá)[4].上皮間質(zhì)轉(zhuǎn)化(Epithelial to mesenchymal transition，EMT)是指上皮細(xì)胞失去極性和細(xì)胞與細(xì)胞之間的連接點(diǎn)，經(jīng)過(guò)細(xì)胞骨架重排、發(fā)生類(lèi)似纖維狀細(xì)胞形態(tài)改變，以此來(lái)增加細(xì)胞遷移和侵襲能力的過(guò)程[5].研究普遍認(rèn)為EMT 的發(fā)生一般與胚胎發(fā)育、組織再生和癌癥轉(zhuǎn)移有密切的聯(lián)系.在腫瘤發(fā)生過(guò)程中，上皮間質(zhì)轉(zhuǎn)化使得分化的上皮腫瘤細(xì)胞變?yōu)橛羞w移和侵襲能力的間質(zhì)腫瘤細(xì)胞，從而使良性腫瘤細(xì)胞浸潤(rùn)周?chē)＝M織，進(jìn)一步使腫瘤細(xì)胞發(fā)生全身性擴(kuò)散和轉(zhuǎn)移[6].已有研究表明，EMT在乳腺癌的轉(zhuǎn)移過(guò)程中發(fā)揮著至關(guān)重要的作用[7].FOXM1參與了腫瘤細(xì)胞的增殖、EMT等過(guò)程的調(diào)控：例如，在胰腺癌細(xì)胞中，高表達(dá)FOXM1B能增強(qiáng)細(xì)胞的生長(zhǎng)能力、集落生成和細(xì)胞遷移能力，并導(dǎo)致細(xì)胞間質(zhì)表型標(biāo)志物表達(dá)上調(diào)和上皮表型標(biāo)志物表達(dá)下降[8]；本實(shí)驗(yàn)室研究也表明FOXM1B能夠促進(jìn)乳腺癌的EMT進(jìn)程，且FOXM1B 的表達(dá)水平與細(xì)胞的上皮和間質(zhì)的特性以及細(xì)胞遷移能力呈正相關(guān)性[6].另一方面，已有研究發(fā)現(xiàn)FOXM1C受Raf/ MEK/ MAPK 信號(hào)通路調(diào)節(jié)，并通過(guò)影響G2/M進(jìn)程從而對(duì)細(xì)胞周期進(jìn)行調(diào)節(jié)[9].同時(shí)，F(xiàn)OXM1C還通過(guò)刺激BMI-1表達(dá)來(lái)抑制氧化應(yīng)激引起的衰老和細(xì)胞增殖[10].目前，腫瘤細(xì)胞中FOXM1B和FOXM1C這兩種FOXM1的剪接異構(gòu)體在功能和定位上是否存在差異還不十分清楚.因此本研究以乳腺癌細(xì)胞的EMT過(guò)程為研究模型，探究FOXM1B和FOXM1C兩種剪接異構(gòu)體在不同表型的乳腺癌細(xì)胞中的定位以及在EMT過(guò)程中是否存在著功能差別.

1 材料與方法

1.1 細(xì)胞培養(yǎng)

人乳腺癌細(xì)胞系MCF-7和MDA-MB-231均購(gòu)置于中國(guó)科學(xué)院細(xì)胞庫(kù)，按ATCC的細(xì)胞培養(yǎng)方法進(jìn)行培養(yǎng).

1.2 細(xì)胞總RNA提取、RT-PCR檢測(cè)相關(guān)基因表達(dá)

RNA的提取按照Total RNA KitI試劑盒(Omega,USA)進(jìn)行，運(yùn)用M-MLV逆轉(zhuǎn)錄酶(Invitrogen,USA)將RNA反轉(zhuǎn)錄為cDNA.PCR檢測(cè)所用引物序列見(jiàn)表1．

表1 PCR引物序列和反應(yīng)條件Tab.1 Primer sequences and reaction conditions of polymerase chain reaction

1.3 質(zhì)?？寺?/p>

以pcDNA3-HA-FOXM1B/C-V5質(zhì)粒(香港大學(xué)生物化學(xué)系提供)為模板，PCR擴(kuò)增得到FOXM1B/C全長(zhǎng)的cDNA序列，將其克隆到pEGFP-C2載體上構(gòu)建pEGFP-FOXM1B/C融合蛋白表達(dá)質(zhì)粒.以pEGFP-C2為模板，PCR擴(kuò)增得到EGFP全長(zhǎng)的cDNA序列，將其克隆到pcDNA3-HA-FOXM1BC-V5質(zhì)粒上構(gòu)建pcDNA3-EGFP-HA-FOXM1B-V5融合蛋白表達(dá)質(zhì)粒.所構(gòu)建的質(zhì)粒均經(jīng)酶切和測(cè)序鑒定.其所用引物見(jiàn)表2．

表2 PCR引物序列和反應(yīng)條件

Tab.2 Primer sequences and reaction conditions of polymerase chain reaction

引物名稱(chēng)序列(5'-3')退火溫度/℃hFOXM1-S:GGAATTCATGAAAGCTAGCCCCCGThFOXM1-AS:CGGGATCCCTACTGTAGCTCAGGAAT58EGFP-S:CCCAAGCTTATGGTGAGCAAGGGCGAGEGFP-AS:CCGGAATTCCGGCCGGACTTGTACAGC58

1.4 細(xì)胞轉(zhuǎn)染與檢測(cè)

轉(zhuǎn)染前一天接種1×10～6細(xì)胞于10 cm 培養(yǎng)皿中，待細(xì)胞鋪滿率達(dá) 80%～90%時(shí)，按Lipofectamine2000說(shuō)明進(jìn)行轉(zhuǎn)染實(shí)驗(yàn)，于48 h后進(jìn)行收樣檢測(cè)．

1.5 Transwell 實(shí)驗(yàn)

取對(duì)數(shù)生長(zhǎng)的細(xì)胞，胰酶消化，細(xì)胞數(shù)為1×105/孔，上室用1% FBS的 DMEM 培養(yǎng)基培養(yǎng)，下室用5% FBS 的 DMEM 細(xì)胞培養(yǎng)基，37 ℃常規(guī)細(xì)胞培養(yǎng) 20～24 h 后，用0.1%的結(jié)晶紫進(jìn)行細(xì)胞染色后，置于顯微鏡下拍照，隨機(jī)取 3～5 個(gè)視野，用 image J 軟件對(duì)照片進(jìn)行數(shù)據(jù)分析.

1.6 統(tǒng)計(jì)分析

所有結(jié)果均采用SPSS20.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析，數(shù)據(jù)以X±S表示，兩組間比較采用t-test，P<0.05具有統(tǒng)計(jì)學(xué)意義.

2 結(jié) 果

2.1 FOXM1在間質(zhì)型乳腺癌細(xì)胞的表達(dá)高于上皮型細(xì)胞

顯微鏡下觀察，MCF-7細(xì)胞呈扁平不規(guī)則多角形， E-cadherin表達(dá)量高于MDA-MB-231細(xì)胞，而MDA-MB-231細(xì)胞細(xì)長(zhǎng)如梭形，Vimentin表達(dá)量高于MCF-7細(xì)胞(圖1(a)～(c)).同時(shí)通過(guò)Transwell實(shí)驗(yàn)發(fā)現(xiàn) MDA-MB-231細(xì)胞的侵襲和轉(zhuǎn)移能力明顯強(qiáng)于MCF-7細(xì)胞(圖1(d)(e)).由此可以看出，MCF-7和MDA-MB-231雖同屬于乳腺癌細(xì)胞，但是兩株細(xì)胞的細(xì)胞形態(tài)與基因表達(dá)存在著明顯的不同.研究結(jié)果表明MDA-MB-231屬于間質(zhì)型細(xì)胞類(lèi)型而MCF-7屬于上皮型細(xì)胞類(lèi)型.通過(guò)對(duì)FOXM1的檢測(cè)發(fā)現(xiàn)，兩株細(xì)胞中FOXM1B的mRNA水平無(wú)明顯差別，而MCF-7細(xì)胞中的FOXM1C mRNA水平略高于MDA-MB-231細(xì)胞，但MDA-MB-231細(xì)胞中的FOXM1蛋白表達(dá)明顯高于MCF-7細(xì)胞(圖1(b),(c)).推測(cè)出現(xiàn)這種現(xiàn)象的原因，雖然FOXM1的不同剪接體的mRNA表達(dá)量在這兩株細(xì)胞中存在差異，但實(shí)驗(yàn)中所用的FOXM1抗體無(wú)法區(qū)分兩種剪接異構(gòu)體蛋白，預(yù)示兩種剪接異構(gòu)體所產(chǎn)生蛋白的穩(wěn)定性在這兩株細(xì)胞中不同.

圖1 FOXM1在不同乳腺癌細(xì)胞內(nèi)的表達(dá)

2.2 質(zhì)粒構(gòu)建

如圖 2(a)所示，分別構(gòu)建pEGFP-FOXM1BC,pcDNA3-EGFP-HA-FOXM1BC-V5 4個(gè)質(zhì)粒，經(jīng)酶切鑒定，測(cè)序鑒定正確(圖 2(b)).

(a) 4個(gè)質(zhì)粒的質(zhì)粒圖譜

(b) 4個(gè)質(zhì)粒的克隆鑒定瓊脂糖電泳圖

2.3 重組質(zhì)粒在真核細(xì)胞內(nèi)的表達(dá)

將所構(gòu)建的重組質(zhì)粒pEGFP-FOXM1BC,pcDNA3-EGFP-HA-FOXM1BC-V5分別轉(zhuǎn)染MDA-MB-231和MCF-7細(xì)胞，WB檢測(cè)蛋白表達(dá)，結(jié)果表明FOXM1B在MDA-MB-231細(xì)胞中的表達(dá)水平較高，而FOXM1C在MCF7細(xì)胞中的表達(dá)水平較高(圖3(a)).24 h后熒光倒置顯微鏡觀測(cè)顯示pEGFP- FOXM1B在MDA-MB-231細(xì)胞中的表達(dá)量高于pEGFP- FOXM1C(圖3(b))，而pcDNA3-EGFP-HA-FOXM1B-V5在MCF-7細(xì)胞中表達(dá)略低于pcDNA3-EGFP-HA-FOXM1C-V5(圖3(d))，結(jié)果與蛋白檢測(cè)結(jié)果一致.并且FOXM1B主要在細(xì)胞核表達(dá)，而FOXM1C在細(xì)胞核和細(xì)胞質(zhì)中均有表達(dá)(圖3(b)～(e)).

2.4 FOXM1B促進(jìn)上皮間質(zhì)轉(zhuǎn)化的發(fā)生

在上皮型細(xì)胞MCF-7內(nèi)高表達(dá)FOXM1B，收集樣本進(jìn)行相關(guān)檢測(cè)，結(jié)果顯示上皮型標(biāo)志物E-cadherin表達(dá)水平顯著下調(diào)，而間質(zhì)型標(biāo)志物Vimentin表達(dá)水平顯著上調(diào)(圖 4(a),(b)).Transwell實(shí)驗(yàn)同時(shí)證明FOXM1B高表達(dá)的MCF-7細(xì)胞的遷移能力明顯高于對(duì)照組(圖4(c),(d)).這些結(jié)果表明FOXM1B的高表達(dá)能夠誘導(dǎo)MCF-7細(xì)胞向間質(zhì)型細(xì)胞轉(zhuǎn)化，使細(xì)胞呈現(xiàn)間質(zhì)型細(xì)胞特性，促使EMT的發(fā)生.

2.5 FOXM1C抑制上皮間質(zhì)轉(zhuǎn)化的發(fā)生

在間質(zhì)型細(xì)胞MDA-MB-231內(nèi)高表達(dá)FOXM1C，收集樣本進(jìn)行相關(guān)檢測(cè)，結(jié)果顯示間質(zhì)型標(biāo)志物Vimentin水平顯著下調(diào)，而上皮型標(biāo)志物E-cadherin顯著上調(diào)(圖 5(a),(b)).同時(shí)Transwell實(shí)驗(yàn)表明高表達(dá)FOXM1C的MDA-MB-231細(xì)胞的遷移能力比對(duì)照組低(圖5(c),(d)).以上結(jié)果表明高表達(dá)FOXM1C能夠誘導(dǎo)MDA-MB-231向上皮型細(xì)胞轉(zhuǎn)化，使細(xì)胞呈現(xiàn)上皮型細(xì)胞特性，抑制EMT的發(fā)生.

圖3 重組質(zhì)粒在乳腺癌細(xì)胞內(nèi)的表達(dá)

圖4 高表達(dá)FOXM1B的MCF-7細(xì)胞出現(xiàn)間質(zhì)型特性

圖5 高表達(dá)FOXM1C的MDA-MB-231細(xì)胞出現(xiàn)上皮型特性

3 討 論

本文將構(gòu)建好的FOXM1B和FOXM1C真核表達(dá)質(zhì)粒轉(zhuǎn)入乳腺癌細(xì)胞，發(fā)現(xiàn)高表達(dá)FOXM1B 的MCF-7細(xì)胞上皮型標(biāo)志物E-cadherin表達(dá)水平顯著下調(diào)，而間質(zhì)型標(biāo)志物Vimentin表達(dá)水平顯著上調(diào).Transwell實(shí)驗(yàn)同時(shí)證明FOXM1B高表達(dá)的MCF-7細(xì)胞的遷移能力明顯高于對(duì)照組，由此表明FOXM1B的高表達(dá)能夠誘導(dǎo)MCF-7細(xì)胞向間質(zhì)型細(xì)胞轉(zhuǎn)化，使細(xì)胞呈現(xiàn)間質(zhì)型細(xì)胞特性，說(shuō)明FOXM1B能夠促進(jìn)EMT的發(fā)生.Park H J等人的研究也發(fā)現(xiàn)FOXM1B是腫瘤發(fā)生轉(zhuǎn)移的活化劑，通過(guò)激活A(yù)kt-SNAIL1通路并刺激Stathmin，賴(lài)氨酰氧化酶，進(jìn)而調(diào)節(jié)癌癥轉(zhuǎn)移相關(guān)基因的表達(dá)[13].由此可見(jiàn)，F(xiàn)OXM1B可受多種信號(hào)通路調(diào)節(jié)，激活癌細(xì)胞轉(zhuǎn)移的相關(guān)標(biāo)志性基因，進(jìn)而激活EMT過(guò)程.而高表達(dá)FOXM1C的MDA-MB-231細(xì)胞顯示其間質(zhì)型標(biāo)志物Vimentin水平顯著下調(diào)，而上皮型標(biāo)志物E-cadherin顯著上調(diào).同時(shí)Transwell實(shí)驗(yàn)表明高表達(dá)FOXM1C的MDA-MB-231細(xì)胞的遷移能力比對(duì)照組低，由此表明高表達(dá)FOXM1C能夠誘導(dǎo)MDA-MB-231向上皮型細(xì)胞轉(zhuǎn)化，使細(xì)胞呈現(xiàn)上皮型細(xì)胞特性，說(shuō)明FOXM1C能抑制EMT的發(fā)生.Wierstra I等人研究發(fā)現(xiàn)FOXM1C可直接結(jié)合到上皮表型重要標(biāo)志物E-cadherin的啟動(dòng)子序列上直接調(diào)節(jié)E-cadherin基因的表達(dá)，進(jìn)而抑制EMT的發(fā)生[14].也有研究表明細(xì)胞可通過(guò)Raf / MEK/ MAPK信號(hào)刺激FOXM1C發(fā)生核轉(zhuǎn)位，從而激活抑癌基因的表達(dá)，實(shí)現(xiàn)抑制EMT的作用[9].

綜上所述，F(xiàn)OXM1B在間質(zhì)細(xì)胞中高表達(dá)，同時(shí)促進(jìn)EMT的發(fā)生，而FOXM1C在上皮型細(xì)胞中高表達(dá)，同時(shí)抑制EMT的發(fā)生.可以看出FOXM1B與FOXM1C作為FOXM1的剪接體具有完全相反的功能.因此，作為EMT的關(guān)鍵調(diào)控因子，F(xiàn)OXM1的不同剪接異構(gòu)體FOXM1B和FOXM1C有望為乳腺癌的治療提供潛在的作用靶點(diǎn).

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A Preliminary Study of Transcription Factor FOXM1 Isoforms in Breast Cancer EMT Process

TAN Yong-jun?,CHEN Han-xiao

(College of Biology, Hunan Univ, Changsha, Hunan 410082, China)

To explore the impact of different transcription factor FOXM1 isoforms in breast cancer EMT process, the eukaryotic expression plasmids for two FOXM1 isoforms, FOXM1B-EGFP and FOXM1C-EGFP, were constructed and transfected into breast cancer cells. The expression levels of FOXM1 isoforms and EMT related genes in the cells were detected with RT-PCR and Western blot. The migration ability of the cells overexpressing the FOXM1 isoforms was measured with the transwell test. The FOXM1B-EGFP and FOXM1C-EGFP eukaryotic expression plasmids were successfully constructed. We found that the levels of exogenous FOXM1B in mesenchymal cells were higher than those in epithelial cells, and it was mainly located in the nucleus. The high levels of FOXM1B expression significantly stimulated the invasion of breast cancer cells and EMT process. The levels of exogenous FOXM1C in epithelial cells were higher than those in mesenchymal cells, and they were expressed in both the nucleus and the cytoplasm. The high levels of FOXM1C expression inhibited the invasion of breast cancer cells and EMT process. FOXM1B was located mainly in the nucleus of cells and FOXM1C was expressed in both the nucleus and the cytoplasm of cells. The research has indicated that FOXM1B and FOXM1C play different roles in the process of EMT of breast cancer cells.

cell adhesion; FOXM1B；FOXM1C；breast cancer；EMT

2015-03-11

國(guó)家自然科學(xué)基金資助項(xiàng)目(81472718)，National Natural Science Foundation of China(81472718)

譚擁軍(1967-)，男，湖南長(zhǎng)沙人，湖南大學(xué)教授

?通訊聯(lián)系人，E-mail:yitan@hnu.edu.cn

1674-2974(2015)12-0100-07

Q279

A