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转录因子FOXM1剪接异构体在乳腺癌EMT过程中的初步研究

2016-03-05谭拥军陈含笑��

湖南大学学报·自然科学版 2015年12期
关键词:乳腺癌

谭拥军++陈含笑��

摘 要:探究转录因子FOXM1不同的剪接异构体对乳腺癌EMT过程中的影响.采用基因工程方法分别构建了表达FOXM1B-EGFP和FOXM1C-EGFP两种 FOXM1剪接异构体真核表达质粒,并将其转染进乳腺癌细胞,采用RT-PCR和Western印迹检测细胞样本中FOXM1剪接异构体的表达和EMT相关基因表达,同时采用transwell检测高表达不同FOXM1剪接异构体细胞的侵袭和迁移能力.成功构建了FOXM1B-EGFP和FOXM1C-EGFP真核表达质粒.外源FOXM1B在乳腺癌间质型细胞的表达高于上皮型细胞,并主要存在于细胞核内,且高表达FOXM1B 能够显著促进乳腺癌细胞的侵袭和EMT过程.外源FOXM1C在乳腺癌上皮型细胞中的表达高于间质型细胞,并且在细胞核和细胞质中均有表达,高表达FOXM1C能够显著抑制细胞的侵袭和EMT过程.实验结果表明,在乳腺癌细胞中,FOXM1B主要存在于细胞核内,FOXM1C在细胞核和细胞质中均有表达.本研究预示FOXM1B和FOXM1C对乳腺癌细胞EMT过程发挥不同的影响.

关键词:细胞粘附;FOXM1B;FOXM1C;乳腺癌; EMT

中图分类号:Q279 文献标识码:A

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)

Abstract: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.

Key words:cell adhesion; FOXM1B;FOXM1C;breast cancer;EMT

FOXM1(Forkhead box protein M1)是叉头框(Forkhead box,Fox)转录因子家族中的成员,又称为Trident,HFH-11,Win,MPP-2等,定位于 12p13-3 号染色体,由十个外显子组成[ 1].FOXM1的蛋白表达存在着A,B 和C 3种剪接异构体:FOXM1A 比FOXM1B多两个外显子,不具有转录活性,在细胞中表达很低;FOXM1C比FOXM1B多一个外显子,和FOXM1B一样具有转录活性,且在肿瘤组织中有高表达,但在某些方面具有不同的生物学功能[ 2-3];FOXM1B主要在癌细胞中高表达,而FOXM1C在正常细胞和癌细胞中均有高表达[ 4].上皮间质转化(Epithelial to mesenchymal transition,EMT)是指上皮细胞失去极性和细胞与细胞之间的连接点,经过细胞骨架重排、发生类似纤维状细胞形态改变,以此来增加细胞迁移和侵袭能力的过程[ 5].研究普遍认为EMT 的发生一般与胚胎发育、组织再生和癌症转移有密切的联系.在肿瘤发生过程中,上皮间质转化使得分化的上皮肿瘤细胞变为有迁移和侵袭能力的间质肿瘤细胞,从而使良性肿瘤细胞浸润周围正常组织,进一步使肿瘤细胞发生全身性扩散和转移[ 6].已有研究表明,EMT在乳腺癌的转移过程中发挥着至关重要的作用[ 7].FOXM1参与了肿瘤细胞的增殖、EMT等过程的调控:例如,在胰腺癌细胞中,高表达FOXM1B能增强细胞的生长能力、集落生成和细胞迁移能力,并导致细胞间质表型标志物表达上调和上皮表型标志物表达下降[ 8];本实验室研究也表明FOXM1B能够促进乳腺癌的EMT进程,且FOXM1B 的表达水平与细胞的上皮和间质的特性以及细胞迁移能力呈正相关性[ 6].另一方面,已有研究发现FOXM1C受Raf/ MEK/ MAPK 信号通路调节,并通过影响G2/M进程从而对细胞周期进行调节[ 9].同时,FOXM1C还通过刺激BMI-1表达来抑制氧化应激引起的衰老和细胞增殖[ 10].目前,肿瘤细胞中FOXM1B和FOXM1C这两种FOXM1的剪接异构体在功能和定位上是否存在差异还不十分清楚.因此本研究以乳腺癌细胞的EMT过程为研究模型,探究FOXM1B和FOXM1C两种剪接异构体在不同表型的乳腺癌细胞中的定位以及在EMT过程中是否存在着功能差别.

1 材料与方法

1.1 细胞培养

人乳腺癌细胞系MCF-7和MDA-MB-231均购置于中国科学院细胞库,按ATCC的细胞培养方法进行培养.

1.2 细胞总RNA提取、RT-PCR检测相关基因表达

RNA的提取按照Total RNA KitI试剂盒(Omega,USA)进行,运用M-MLV逆转录酶(Invitrogen,USA)将RNA反转录为cDNA.PCR检测所用引物序列见表1.

1.3 质粒克隆

以pcDNA3-HA-FOXM1B/C-V5质粒(香港大学生物化学系提供)为模板,PCR扩增得到FOXM1B/C全长的cDNA序列,将其克隆到pEGFP-C2载体上构建pEGFP-FOXM1B/C融合蛋白表达质粒.以pEGFP-C2为模板,PCR扩增得到EGFP全长的cDNA序列,将其克隆到pcDNA3-HA-FOXM1B\\C-V5质粒上构建pcDNA3-EGFP-HA-FOXM1B-V5融合蛋白表达质粒.所构建的质粒均经酶切和测序鉴定.其所用引物见表2.

1.4 细胞转染与检测

转染前一天接种1×10~6细胞于10 cm 培养皿中,待细胞铺满率达 80%~90%时,按Lipofectamine2000说明进行转染实验,于48 h后进行收样检测.

1.5 Transwell 实验

取对数生长的细胞,胰酶消化,细胞数为1×10 5/孔,上室用1% FBS的 DMEM 培养基培养,下室用5% FBS 的 DMEM 细胞培养基,37 ℃常规细胞培养 20~24 h 后,用0.1%的结晶紫进行细胞染色后,置于显微镜下拍照,随机取 3~5 个视野,用 image J 软件对照片进行数据分析.

1.6 统计分析

所有结果均采用SPSS20.0统计软件进行统计分析,数据以X±S表示,两组间比较采用t-test,P<0.05具有统计学意义.

2 结 果

2.1 FOXM1在间质型乳腺癌细胞的表达高于上皮型细胞

显微镜下观察,MCF-7细胞呈扁平不规则多角形, E-cadherin表达量高于MDA-MB-231细胞,而MDA-MB-231细胞细长如梭形,Vimentin表达量高于MCF-7细胞(图1(a)~(c)).同时通过Transwell实验发现 MDA-MB-231细胞的侵袭和转移能力明显强于MCF-7细胞(图1(d)(e)).由此可以看出,MCF-7和MDA-MB-231虽同属于乳腺癌细胞,

但是两株细胞的细胞形态与基因表达存在着明显的不同.研究结果表明MDA-MB-231属于间质型细胞类型而MCF-7属于上皮型细胞类型.通过对FOXM1的检测发现,两株细胞中FOXM1B的mRNA水平无明显差别,而MCF-7细胞中的FOXM1C mRNA水平略高于MDA-MB-231细胞,但MDA-MB-231细胞中的FOXM1蛋白表达明显高于MCF-7细胞(图1(b),(c)).推测出现这种现象的原因,虽然FOXM1的不同剪接体的mRNA表达量在这两株细胞中存在差异,但实验中所用的FOXM1抗体无法区分两种剪接异构体蛋白,预示两种剪接异构体所产生蛋白的稳定性在这两株细胞中不同.

2.2 质粒构建

如图 2(a)所示,分别构建pEGFP-FOXM1B\\C,pcDNA3-EGFP-HA-FOXM1B\\C-V5 4个质粒,经酶切鉴定,测序鉴定正确(图 2(b)).

(a) 4个质粒的质粒图谱

(b) 4个质粒的克隆鉴定琼脂糖电泳图

2.3 重组质粒在真核细胞内的表达

将所构建的重组质粒pEGFP-FOXM1B\\C,pcDNA3-EGFP-HA-FOXM1B\\C-V5分别转染MDA-MB-231和MCF-7细胞,WB检测蛋白表达,结果表明FOXM1B在MDA-MB-231细胞中的表达水平较高,而FOXM1C在MCF7细胞中的表达水平较高(图3(a)).24 h后荧光倒置显微镜观测显示pEGFP- FOXM1B在MDA-MB-231细胞中的表达量高于pEGFP- FOXM1C(图3(b)),而pcDNA3-EGFP-HA-FOXM1B-V5在MCF-7细胞中表达略低于pcDNA3-EGFP-HA-FOXM1C-V5(图3(d)),结果与蛋白检测结果一致.并且FOXM1B主要在细胞核表达,而FOXM1C在细胞核和细胞质中均有表达(图3(b)~(e)).

2.4 FOXM1B促进上皮间质转化的发生

在上皮型细胞MCF-7内高表达FOXM1B,收集样本进行相关检测,结果显示上皮型标志物E-cadherin表达水平显著下调,而间质型标志物Vimentin表达水平显著上调(图 4(a),(b)).Transwell实验同时证明FOXM1B高表达的MCF-7细胞的迁移能力明显高于对照组(图4(c),(d)).这些结果表明FOXM1B的高表达能够诱导MCF-7细胞向间质型细胞转化,使细胞呈现间质型细胞特性,促使EMT的发生.

2.5 FOXM1C抑制上皮间质转化的发生

在间质型细胞MDA-MB-231内高表达FOXM1C,收集样本进行相关检测,结果显示间质型标志物Vimentin水平显著下调,而上皮型标志物E-cadherin显著上调(图 5(a),(b)).同时Transwell实验表明高表达FOXM1C的MDA-MB-231细胞的迁移能力比对照组低(图5(c),(d)).以上结果表明高表达FOXM1C能够诱导MDA-MB-231向上皮型细胞转化,使细胞呈现上皮型细胞特性,抑制EMT的发生.

3 讨 论

本文将构建好的FOXM1B和FOXM1C真核表达质粒转入乳腺癌细胞,发现高表达FOXM1B 的MCF-7细胞上皮型标志物E-cadherin表达水平显著下调,而间质型标志物Vimentin表达水平显著上调.Transwell实验同时证明FOXM1B高表达的MCF-7细胞的迁移能力明显高于对照组,由此表明FOXM1B的高表达能够诱导MCF-7细胞向间质型细胞转化,使细胞呈现间质型细胞特性,说明FOXM1B能够促进EMT的发生.Park H J等人的研究也发现FOXM1B是肿瘤发生转移的活化剂,通过激活Akt-SNAIL1通路并刺激Stathmin,赖氨酰氧化酶,进而调节癌症转移相关基因的表达[ 13].由此可见,FOXM1B可受多种信号通路调节,激活癌细胞转移的相关标志性基因,进而激活EMT过程.而高表达FOXM1C的MDA-MB-231细胞显示其间质型标志物Vimentin水平显著下调,而上皮型标志物E-cadherin显著上调.同时Transwell实验表明高表达FOXM1C的MDA-MB-231细胞的迁移能力比对照组低,由此表明高表达FOXM1C能够诱导MDA-MB-231向上皮型细胞转化,使细胞呈现上皮型细胞特性,说明FOXM1C能抑制EMT的发生.Wierstra I等人研究发现FOXM1C可直接结合到上皮表型重要标志物E-cadherin的启动子序列上直接调节E-cadherin基因的表达,进而抑制EMT的发生[ 14].也有研究表明细胞可通过Raf / MEK/ MAPK信号刺激FOXM1C发生核转位,从而激活抑癌基因的表达,实现抑制EMT的作用[ 9].

综上所述,FOXM1B在间质细胞中高表达,同时促进EMT的发生,而FOXM1C在上皮型细胞中高表达,同时抑制EMT的发生.可以看出FOXM1B与FOXM1C作为FOXM1的剪接体具有完全相反的功能.因此,作为EMT的关键调控因子,FOXM1的不同剪接异构体FOXM1B和FOXM1C有望为乳腺癌的治疗提供潜在的作用靶点.

参考文献

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