尹崇高 李洪利李文通孫永紅張寶剛＊

(山東省濰坊醫(yī)學(xué)院護(hù)理學(xué)院,1科研處醫(yī)學(xué)研究實驗中心,2病理學(xué)教研室,3附屬醫(yī)院病理科,濰坊 261053)

TGF-β1對乳腺癌細(xì)胞系Snail表達(dá)的影響

尹崇高 李洪利1李文通2孫永紅3張寶剛2＊

(山東省濰坊醫(yī)學(xué)院護(hù)理學(xué)院,1科研處醫(yī)學(xué)研究實驗中心,2病理學(xué)教研室,3附屬醫(yī)院病理科,濰坊 261053)

目的 通過 TGF-β1誘導(dǎo)乳腺癌 MCF-7發(fā)生上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)后檢測鋅指轉(zhuǎn)錄因子Snail表達(dá)的改變,探討Snail在EMT及乳腺癌發(fā)生發(fā)展中的作用。方法 常規(guī)培養(yǎng)乳腺癌細(xì)胞株MCF-7后,用 TGF-β1誘導(dǎo)其發(fā)生 EMT,用 Transwell侵襲小室法進(jìn)行細(xì)胞體外侵襲能力檢測;用免疫組織化學(xué)方法及免疫熒光檢測 E-cadherin、Vimentin、Snail的表達(dá);用 real time PCR檢測 E-cadherin、Vimentin 、Snail mRNA 的表達(dá)。結(jié)果 TGF-β1處理72h后的MCF-7細(xì)胞穿透能力明顯增強(qiáng)。E-cadherin蛋白及mRNA表達(dá)減少,Vimentin、Snail蛋白及mRNA表達(dá)增加。結(jié)論E-cadherin、Vimentin是細(xì)胞發(fā)生 EMT的重要生物學(xué)標(biāo)志,Snail可能在轉(zhuǎn)錄水平上調(diào)控 E-cadherin、Vimentin蛋白的表達(dá),Snail在EMT和乳腺癌的發(fā)生發(fā)展中起著重要的作用。

TGF-β1; 乳腺腫瘤; 上皮-間質(zhì)轉(zhuǎn)化; Snail

上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)發(fā)生于多種生理、病理過程,如胚胎發(fā)育、腫瘤轉(zhuǎn)移等,其共同的細(xì)胞學(xué)機(jī)制為細(xì)胞間粘附的喪失與細(xì)胞遷移力的獲得[1]。Snail是一種在EMT過程中居于重要地位的轉(zhuǎn)錄因子。EMT在多種腫瘤細(xì)胞獲得侵襲轉(zhuǎn)移能力中有重要作用[2]。轉(zhuǎn)化生長因子-β1(transforming growth factor-β1,TGF-β1)是細(xì)胞外環(huán)境中重要的細(xì)胞因子,研究表明 TGF-β1能刺激正常犬腎上皮細(xì)胞(MDCK)向間質(zhì)細(xì)胞表型轉(zhuǎn)化[3]。本實驗通過體外研究 TGF-β1對MCF-7遷移能力及Snail表達(dá)的影響,深入探討提高M(jìn)CF-7遷移力及可能的分子機(jī)制。

材料和方法

1. 材料

MCF-7細(xì)胞系由本實驗中心保存提供,TGF-β1購自R&D公司;AMV第一鏈cDNA合成試劑盒購自上海生工生物技術(shù)有限公司;iQ SYBR Green Supermix購自北京元業(yè)伯樂有限公司;RPMI1640培養(yǎng)基為 Hyclone公司產(chǎn)品;E-cadherin、Vimentin、Snail一抗購自 Santa Cruz公司;引物由上海生工生物技術(shù)有限公司合成;標(biāo)記有Cy3的羊抗兔二抗購自北京中杉生物技術(shù)有限公司。

2. 方法

2.1 細(xì)胞培養(yǎng)與處理

乳腺癌細(xì)胞系MCF-7培養(yǎng)在含10%胎牛血清的RPMI1640細(xì)胞培養(yǎng)液中,37℃、5%CO2培養(yǎng)箱中培養(yǎng)。實驗組用 0.2μg/L 的 TGF-β1處理72h,細(xì)胞80%融合時用于實驗。

2.2 細(xì)胞形態(tài)學(xué)觀察

光學(xué)顯微鏡下觀察未處理組及 0.2μg/L的TGF-β1處理72h后細(xì)胞形態(tài)學(xué)的改變。

2.3 Transwell侵襲小室體外侵襲實驗

參照Ries等[4]的方法,取 Transwell小室放入24孔板中,小室濾膜的上方加重組細(xì)胞基底膜(Matrigel)20μg形成基質(zhì)膠層,之后將細(xì)胞制成單細(xì)胞懸液,細(xì)胞以 1×105/cm2密度,每孔 200μl接種于 Transwell膜上,并在 Transwell下室中加入含 0.2μg/L 的 TGF-β1的 RPMI1640 培養(yǎng)基作為趨化劑 ,每孔 600μl。于 37 ℃、5%CO2培育 48h 后 ,吸取 Transwell小室中的培養(yǎng)液,以棉簽輕輕拭去微孔膜上層的細(xì)胞,保留侵襲至微孔膜下表面的細(xì)胞,用冰甲醇固定1min后 HE染色。于高倍鏡(×200)下計數(shù)穿過基質(zhì)膜濾膜底面的細(xì)胞數(shù)。

2.4 免疫細(xì)胞化學(xué)方法檢測細(xì)胞內(nèi) E-cadherin、Vimentin及 Snail的表達(dá)

將未處理組及0.2μg/L的 TGF-β1處理72h后的MCF-7細(xì)胞接種于涂有多聚賴氨酸的玻片上,貼壁后,取出玻片,用預(yù)冷的丙酮固定細(xì)胞5min,PBS沖洗3次,加入稀釋的一抗孵育30min,PBS沖洗3次,加入二抗孵育30min,PBS沖洗3次,加入三抗孵育30min,PBS沖洗3次后DAB顯色。結(jié)果判定參照文獻(xiàn)采用半定量積分法[5]:高倍鏡下連續(xù)觀察5個高倍視野,以陽性細(xì)胞數(shù)所占比例小于5%為0分,5%-25%為1分,26%-50%為2分,51%-75%為3分,大于75%為4分。染色強(qiáng)度:以無染色為0分,淡黃色為1分,黃或深黃色為2分,褐色或棕褐色為3分。兩項積分相乘大于4分為陽性。實驗重復(fù)3次,取其均值作統(tǒng)計學(xué)分析。

2.5 免疫熒光方法檢測細(xì)胞內(nèi) E-cadherin、Vimentin及Snail的表達(dá)

將未處理組及0.2μg/L的 TGF-β1處理72h后的MCF-7細(xì)胞制作細(xì)胞爬片,在甲醇及丙酮(體積比3∶1)新鮮配置的混合冷固定液中固定20min后,用蒸餾水洗2次后,加入 H2O2封閉30min,PBS洗3次每次5min,然后加一抗4℃過夜,PBS洗三次,每次5min,滴加Cy3標(biāo)記的羊抗兔 IgG作為二抗37℃孵育30min,PBS洗三次后,滴加DAPI(復(fù)染細(xì)胞核,呈藍(lán)色熒光)室溫孵育5min。熒光顯微鏡下觀察。用PBS代替一抗作為陰性對照。結(jié)果判定同免疫細(xì)胞化學(xué)采用的半定量積分法。

2.6 real time PCR 檢測細(xì)胞內(nèi) E-cadherin、Vimentin及Snail mRNA的表達(dá)

Trizol法提取細(xì)胞中的總 RNA,逆轉(zhuǎn)錄合成cDNA,real time PCR檢測樣本中的 E-cadherin、Vimentin及Snail cDNA水平,β-actin基因做內(nèi)參照。引 物:Snail(799bp):5’-CCACTATGCCGCGCTCTTT-3 ’ (up),5 ’-TCA GCGGGGACATCCTGA GCA-3’(down);E-cadherin(377bp):5’-A TCCAAAGCCTCAGGTCATAAACA-3’(up),5’-AAGAAACAGCAAGA GCA GCAGAA T-3’(down);Vimentin(690bp):5’-CGC TTC GCC AAC TAC AT-3’(up)5’-AGG GCA TCC ACT TCA CAG-3’(down);β-actin(116bp):5’-TGGATCAGCAAGCAGGAGTA TGACGA GT-3’(up),5’-CGCAAGTTAGGTTTTGTCAAGAAAGGGT-3’(down)。反應(yīng)在 25μl的體系中進(jìn)行,其中 iQ SYBR Green Supermix 12.5μl、10μmol/L 的上下游引物各 1μl、樣本 cDNA2μl(300ng)、RNA 酶滅活水8.5μl。熱循環(huán)條件如下:Snail:94℃30s→(94℃15s→55℃30s→68℃30s40cycle)→68℃5min。E-cadherin:94℃30s→(94℃15s→57℃30s→68℃30s38cycle)→68℃5min。β-actin作為內(nèi)參照同時擴(kuò)增。按公式計算樣本中 Snail、E-cadherin、Vimentin mRNA的相對表達(dá)量。

3.統(tǒng)計學(xué)分析

采用 SPSS13.0軟件分析數(shù)據(jù),不同組細(xì)胞Snail、E-cadherin、Vimentin 蛋白及 mRNA 表達(dá)水平的比較采用獨(dú)立樣本t檢驗。P<0.05為差異有統(tǒng)計學(xué)意義。

結(jié) 果

1.細(xì)胞形態(tài)學(xué)的改變

光學(xué)顯微鏡下觀察 0.2μg/L 的 TGF-β1處理72h后的MCF-7細(xì)胞發(fā)現(xiàn)與未處理組細(xì)胞相比,細(xì)胞突起不同程度的增多,細(xì)胞變得細(xì)長,細(xì)胞融合度降低(見圖1)。

2.免疫細(xì)胞化學(xué)染色及免疫熒光觀察MCF-7細(xì)胞中Snail、E-cadherin及Vimentin蛋白的表達(dá)

Snail主要表達(dá)于MCF-7細(xì)胞的細(xì)胞核中,呈棕黃色顆粒狀(見圖2),免疫熒光顯示紅色(圖3)。0.2μg/L 的 TGF-β1處理 72h 后的 MCF-7 陽性細(xì)胞積分(5-6分)明顯高于未處理組的陽性細(xì)胞積分(1分),差異有統(tǒng)計學(xué)意義。

圖1 TGF-β1處理前后MCF-7細(xì)胞形態(tài)學(xué)的改變 ×200Fig. 1 Morphological changes of TGF-β1treated and untreated MCF-7 cells×200

E-cadherin主要表達(dá)于MCF-7細(xì)胞的細(xì)胞膜及細(xì)胞漿中,呈棕黃色、顆粒狀(見圖2),免疫熒光顯示紅色(圖 3)。0.2μg/L 的 TGF-β1處理 72h后的MCF-7陽性細(xì)胞積分(5-6分)明顯低于未處理組的陽性細(xì)胞數(shù)(2-3分),差異有統(tǒng)計學(xué)意義。

Vimentin主要表達(dá)于 MCF-7細(xì)胞的細(xì)胞漿中,呈棕黃色、顆粒狀(見圖2),免疫熒光顯示紅色(圖 3)。0.2μg/L 的 TGF-β1處理 72h后的 MCF-7陽性細(xì)胞積分(3-4分)明顯高于未處理組的陽性細(xì)胞積分(2分),差異有統(tǒng)計學(xué)意義。

3.TGF-β1增強(qiáng)了MCF-7細(xì)胞的體外侵襲能力

圖2 免疫組化顯示 TGF-β1處理前后細(xì)胞內(nèi)Snail、E-cadherin、Vimentin的改變 ×200圖3 免疫熒光顯示 TGF-β1處理前后細(xì)胞內(nèi)Snail、E-cadherin、Vimentin的改變 ×200Fig.2 Changes of Snail,E-cadherin and Vimentin in cells by immunohistochemistry ×200Fig.3 Changes of Snail,E-cadherin and Vimentin in cells by immunofluorescence ×200

Transwell小室侵襲實驗結(jié)果顯示用 TGF-β1做誘導(dǎo)劑的MCF-7細(xì)胞穿透到濾膜下的細(xì)胞數(shù)顯著增加,未處理組 MCF-7細(xì)胞的穿透數(shù)為94±3個,用 TGF-β1做誘導(dǎo)劑的MCF-7細(xì)胞的穿透數(shù)為176±7個,差異有統(tǒng)計學(xué)意義。TGF-β1顯著促進(jìn)了MCF-7細(xì)胞在體外的侵襲能力。

4. 細(xì)胞中 Snail、E-cadherin及 Vimentin mRNA的表達(dá)

Real-time PCR檢測將未處理組MCF-7細(xì)胞中 E-cadherin、Vimentin、Snail mRNA 與β-actin mRNA的比值分別作為1,與未處理組細(xì)胞相比,TGF-β1做誘導(dǎo)劑的 MCF-7 細(xì)胞 Snail(2.012)、Vimentin(2.125)mRNA 表達(dá)明顯升高 (P<0.05);E-cadherin mRNA表達(dá)明顯降低(0.569)。

討 論

乳腺癌是女性最常見的惡性腫瘤之一,其發(fā)病率逐年增加,盡管臨床研究已經(jīng)取得了較大的進(jìn)步,但是其轉(zhuǎn)移及復(fù)發(fā)率仍然很高,乳腺癌預(yù)后已成為臨床研究的熱點(diǎn)問題[6]。EMT指上皮細(xì)胞在特定生理和病理情況下向間充質(zhì)細(xì)胞轉(zhuǎn)化的想象。在胚胎發(fā)育、組織成形、傷口愈合、慢性炎癥和多種纖維化疾病中,EMT發(fā)揮了重要作用[7]。腫瘤細(xì)胞在晚期常在細(xì)胞因子(TGF-為最常見)等作用下發(fā)生EMT而發(fā)生浸潤及轉(zhuǎn)移[8]。EMT主要的表現(xiàn)包括細(xì)胞間失去粘附和極性,骨架重塑,酶解基底膜發(fā)生轉(zhuǎn)移。本實驗形態(tài)學(xué)結(jié)果顯示細(xì)胞突起不同程度的增多,細(xì)胞變得細(xì)長,細(xì)胞融合度降低,而免疫組化及免疫熒光結(jié)果顯示 TGF-1作用后能證明細(xì)胞發(fā)生EMT的相關(guān)基因如 E-cadherin明顯降低、而vimentin明顯升高,表明TGF-1能有效的促進(jìn)乳腺癌細(xì)胞發(fā)生EMT。

TGF-是一類多功能的細(xì)胞因子,能以自分泌、旁分泌方式調(diào)節(jié)多種細(xì)胞的生物學(xué)功能,包括抑制上皮細(xì)胞、免疫細(xì)胞和造血細(xì)胞的增殖,誘導(dǎo)血管生成。目前國外研究發(fā)現(xiàn) ,TGF-[9]、NF-κB、Ras/MAPK等所介導(dǎo)的信號通路能誘導(dǎo)Snail表達(dá),而糖原合成酶激酶-3則能通過抑制 NF-κB而抑制Snail表達(dá)[10]。Snail是存在于果蠅、鼠、人等生物體內(nèi)的一種堿性螺旋-環(huán)-螺旋轉(zhuǎn)錄因子,包括三個典型和一個非典型的鋅指結(jié)構(gòu)區(qū)的鋅指結(jié)構(gòu),屬于鋅指蛋白家族成員。在胚胎發(fā)育期間,Snail表達(dá)于原腸胚兩側(cè)細(xì)胞并參與了其發(fā)生EMT,進(jìn)而遷移形成中胚層的過程,且能抑制外胚層來源基因的表達(dá)[11]。既往的研究表明:轉(zhuǎn)錄因子Snail高表達(dá)于乳腺癌[12]、胃癌[13]、肝癌[14]、結(jié)腸癌[15]中 ,并與乳腺癌組織學(xué)分級、淋巴結(jié)轉(zhuǎn)移密切相關(guān)。Snail能作用于啟動子上 E-boxes連接基序,直接抑制 E-鈣粘素等上皮粘附位點(diǎn)成分轉(zhuǎn)錄,使細(xì)胞間粘附紊亂。本實驗用 TGF-1誘導(dǎo)了MCF-7細(xì)胞發(fā)生EMT,發(fā)生 EMT的MCF-7細(xì)胞 Snail表達(dá)明顯增強(qiáng),表明Snail在 TGF-1誘導(dǎo)乳腺癌細(xì)胞發(fā)生 EMT中起著重要的作用。

本研究用 0.2μg/L 的 TGF-1處理 MCF-7細(xì)胞72h后即可見MCF-7細(xì)胞形態(tài)發(fā)生了明顯變化,表現(xiàn)為細(xì)胞變得細(xì)長,偽足增多;同時 Vimentin,Snail蛋白及mRNA表達(dá)明顯增高而E-cadherin蛋白、mRNA表達(dá)降低;Transwell小室侵襲實驗提示細(xì)胞轉(zhuǎn)移潛能明顯增強(qiáng)。以上結(jié)果表明:TGF-1可能是通過調(diào)控轉(zhuǎn)錄因子Snail的表達(dá)使細(xì)胞發(fā)生了表型的轉(zhuǎn)化以及增強(qiáng)轉(zhuǎn)移潛能。

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TGF-β1affect expression of snail in breast cancer

Yin Chonggao,Li Hongli1,Li Wentong2,Sun Yonghong3,Zhang Baogang2＊

(Department of N ursing;1Medicine Research Ex periment Center of Science Research;2Department ofPathology,Weif ang Medical College;

Objective To detect the expression of the zinc finger transcription factor snail after TGF-β1-induced epithelial-mesenchymal transition(EMT),and investigate the role of snail in EMT and the development of breast cancer. Methods TGF-β1induced EMT in routinely cultured breast cancer cell line MCF-7. The invasive and metastatic capacity was evaluated with transwell migration assay.The expressions of E-cadherin,vimentin and snail protein and mRNA were investigated by real time PCR,immunohistochemistry and immunofluorescence. Results TGF-β1significantly decreased the expressions of E-cadherin protein and mRNA,but increased the expressions of Vimentin and snail protein and mRNA.The cellular invasion assay indicated that TGF-β1drastically enhanced the invasive potential of MCF-7. Conclusion E-cadherin and Vimentin are important biological markers of EMT.Snail may regulate the expressions of E-cadherin and Vimentin protein at the transcriptional level.Snail plays an important role in EMT and the development of breast cancer.

TGF-β1; Breast tumor; Epithelial-mesenchymal transition; Snail

R329.24;Q291

A

10.3870/zgzzhx.2011.02.0043Department ofPathology,af f iliated hospital of Weif ang Medical College,Weif ang261053,China)

2010-09-10

2011-04-01

國家自然科學(xué)基金(81072068),山東省優(yōu)秀中青年科學(xué)家科研獎勵基金(BS2010yy071),山東省濰坊醫(yī)學(xué)院青年教師啟動基金(KQ07037)

尹崇高,男(1979年),漢族,講師。

＊通訊作者(To whom correspondence should be addressed)