蔡娟娟，崔艷梅，丁彥青，廖雯婷

(南方醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院病理系,廣州 510515)

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FOXC2對結(jié)直腸癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化及侵襲能力的影響*

蔡娟娟，崔艷梅，丁彥青，廖雯婷△

(南方醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院病理系,廣州 510515)

[摘要]目的明確叉頭框C2(FOXC2)在結(jié)直腸癌細(xì)胞侵襲遷移中的作用。方法采用逆轉(zhuǎn)錄病毒感染的方法建立FOXC2穩(wěn)定過表達(dá)的結(jié)直腸癌細(xì)胞株(SW480/FOXC2)及空載體對照細(xì)胞株(SW480/pBabe)，顯微鏡下觀察結(jié)直腸癌細(xì)胞形態(tài)改變。Western blot及免疫熒光法檢測FOXC2過表達(dá)細(xì)胞株及對照細(xì)胞株中E-cadherin、Vimentin、N-cadherin的表達(dá)情況；Transwell侵襲小室實驗檢測結(jié)直腸癌細(xì)胞遷移能力的改變。結(jié)果過表達(dá)后SW480細(xì)胞的形態(tài)發(fā)生了明顯的變化，從原來典型的上皮細(xì)胞形狀變成長梭形，類似于成纖維細(xì)胞的形態(tài)；Western Blot及免疫熒光檢測結(jié)果顯示，F(xiàn)OXC2過表達(dá)后上皮分子標(biāo)志物E-cadherin表達(dá)明顯下調(diào)，而間質(zhì)分子標(biāo)志物Vimentin及N-cadherin表達(dá)顯著上調(diào)；Transwell侵襲實驗結(jié)果顯示，F(xiàn)OXC2過表達(dá)后結(jié)直腸癌細(xì)胞侵襲潛能明顯增強(qiáng)。結(jié)論FOXC2過表達(dá)能誘導(dǎo)結(jié)直腸癌細(xì)胞SW480發(fā)生上皮-間質(zhì)轉(zhuǎn)化并增強(qiáng)其侵襲能力。

[關(guān)鍵詞]結(jié)直腸腫瘤;叉頭框C2;上皮-間質(zhì)轉(zhuǎn)化;侵襲

結(jié)直腸癌是全球發(fā)病率最高的惡性腫瘤之一，全球每年新發(fā)病例已增至100萬，其發(fā)病率以4.2%的速度螺旋遞增[1-2]。轉(zhuǎn)移是導(dǎo)致結(jié)直腸癌治愈率低、死亡率高的主要原因[3-4]。上皮-間質(zhì)轉(zhuǎn)化是腫瘤轉(zhuǎn)移的重要步驟，使細(xì)胞失去上皮細(xì)胞表型獲得間皮細(xì)胞表型，促使細(xì)胞轉(zhuǎn)移至鄰近組織和遠(yuǎn)端器官，引起細(xì)胞的侵襲轉(zhuǎn)移[5-6]。叉頭框C2(forkhead box C2,FOXC2)又名間充質(zhì)叉頭框1(mesenchyme forkhead 1,MHF1)，屬于叉頭框轉(zhuǎn)錄因子超家族成員，最初發(fā)現(xiàn)于小鼠腦組織[7]。人類FOXC2基因定位于16q24.3，其編碼的蛋白參與調(diào)節(jié)包細(xì)胞分化、代謝、發(fā)育、增殖和凋亡等不同的細(xì)胞過程[8]。近年來的研究表明，F(xiàn)OXC2在乳腺癌、胃癌、非小細(xì)胞癌和食管癌中高表達(dá)，其異常高表達(dá)與腫瘤的生長、侵襲和轉(zhuǎn)移能力密切相關(guān)[9-14]。然而，F(xiàn)OXC2在結(jié)直腸癌侵襲轉(zhuǎn)移中的作用及分子機(jī)制尚未研究清楚。本研究旨在探討FOXC2對結(jié)直腸癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化及侵襲遷移潛能的影響。

1材料與方法

1.1材料

1.1.1細(xì)胞株人結(jié)腸癌細(xì)胞株SW480，人胚腎細(xì)胞株293Ft為南方醫(yī)科大學(xué)病理學(xué)實驗室自存。

1.1.2實驗試劑胎牛血清、RPMI-1640購自美國Gibico公司，PVDF膜購自美國Millipore公司，Transwell小室購自美國BD公司，E-cadherin、Vimentin、N-cadherin抗體購自美國Bioworld公司，兔二抗及鼠二抗購自美國Thermo公司，α-Tubulin購自北京銳抗公司。

1.2方法

1.2.1構(gòu)建穩(wěn)定細(xì)胞株(1)培養(yǎng)并傳代病毒細(xì)胞293Ft細(xì)胞，轉(zhuǎn)染前24 h，取對數(shù)生長期細(xì)胞，胰酶消化后，按每孔5×105個接種于6孔板中。當(dāng)細(xì)胞生長成80%時進(jìn)行轉(zhuǎn)染。(2)將重組質(zhì)粒及陰性對照質(zhì)粒各25 μg，慢病毒包裝質(zhì)粒25 μg分別加入EP管中，依次加入Gibico H2O至150 μL，加入CaCl2250 μL，最后加入200 μL緩沖液(2×HBS，pH 7.0)，室溫靜置25 min后加入293Ft細(xì)胞中，隨后加入氯喹30 μL。(3)24 h后，收集細(xì)胞培養(yǎng)上清液，用0.45 μm孔徑的濾器(Millipore公司產(chǎn)品)過濾，收集濾液，按4 μg/mL加入polybrene，混勻后，加至提前1 d接種于6孔細(xì)胞培養(yǎng)板中的待轉(zhuǎn)染細(xì)胞(4×105個/孔)上，每隔4 h收集一次病毒上清液并加入待轉(zhuǎn)染細(xì)胞中，連續(xù)感染3次。(4)感染結(jié)束后24～48 h，用含1 μg/mL嘌呤霉素的培養(yǎng)基篩選陽性克隆，連續(xù)篩選3 d，每天換液。(5)收集以上細(xì)胞株的RNA和蛋白，用熒光定量PCR和Western blot方法鑒定。

1.2.2Western blot蛋白用10.5%SDS-PAGE凝膠進(jìn)行電泳分離，濕法轉(zhuǎn)印至PVDF膜上，轉(zhuǎn)膜結(jié)束后兔抗人FOXC2抗體，E-cadherin、Vimentin、N-cadherin單克隆抗體孵育過夜，次日加入辣根過樣化物酶標(biāo)記的二抗，于室溫孵育45 min后，洗脫抗體，常規(guī)顯色發(fā)光。

1.2.3免疫熒光實驗細(xì)胞轉(zhuǎn)染后24 h消化計數(shù)，爬片，漂洗，固定，再漂洗，加一抗過夜，次日加入帶熒光二抗，避光反應(yīng)1 h，漂洗，染色，激光共聚焦顯微鏡下觀察。

1.2.4Transwell遷移實驗細(xì)胞轉(zhuǎn)染后24 h消化計數(shù)，加入放置了Transwell小室的24孔板中，上層小室加入含5%胎牛血清的培養(yǎng)基，下層小室加入含20%胎牛血清的培養(yǎng)基，孵育48 h，取出，固定，染色，顯微鏡下隨機(jī)選取視野拍照計數(shù)。

1.3統(tǒng)計學(xué)處理采用SPSS21.0統(tǒng)計軟件進(jìn)行分析，兩獨立樣本t檢驗，以P<0.05為差異有統(tǒng)計學(xué)意義。

2結(jié)果

2.1FOXC2過表達(dá)后結(jié)直腸癌細(xì)胞形態(tài)變化FOXC2穩(wěn)定過表達(dá)后，SW480細(xì)胞的形態(tài)發(fā)生了明顯的變化，從原來典型的上皮細(xì)胞形狀變成長梭形，類似于成纖維細(xì)胞的形態(tài)。見圖1。

2.2FOXC2過表達(dá)后上皮-間質(zhì)轉(zhuǎn)化標(biāo)志物的變化Western blot實驗顯示，與對照組相比，F(xiàn)OXC2過表達(dá)后，上皮標(biāo)記物E-cadherin的表達(dá)下調(diào)，而間質(zhì)標(biāo)記物Vimentin，N-cadherin表達(dá)上調(diào)(圖2)。免疫熒光實驗顯示，與對照組相比，F(xiàn)OXC2過表達(dá)后，上皮標(biāo)記物E-cadherin的表達(dá)強(qiáng)度下調(diào)，而間質(zhì)標(biāo)記物Vimentin、N-cadherin表達(dá)強(qiáng)度上調(diào)(圖3)。

圖1　FOXC2過表達(dá)SW480的形態(tài)學(xué)變化

圖2　Western blot檢測FOXC2過表達(dá)細(xì)胞株與對照組細(xì)胞株中上皮-間質(zhì)轉(zhuǎn)化標(biāo)志物的表達(dá)

2.3FOXC2過表達(dá)后結(jié)直腸癌細(xì)胞侵襲遷移潛能的變化Transwell遷移實驗結(jié)果顯示，與對照組相比，結(jié)直腸癌SW480細(xì)胞FOXC2過表達(dá)后遷移的細(xì)胞明顯增多，差異有統(tǒng)計學(xué)意義(P<0.01)。見圖4。

圖3　免疫熒光法檢測FOXC2過表達(dá)細(xì)胞株及對照細(xì)胞株中E-cadherin、Vimentin、N-cadherin的表達(dá)情況(×60)

圖4　Transwell侵襲小室實驗檢測結(jié)直腸癌細(xì)胞遷移能力的改變(×20)

3討論

腫瘤轉(zhuǎn)移是一個多步驟、多階段的復(fù)雜過程，包括腫瘤微環(huán)境的改變、細(xì)胞遷移、細(xì)胞運(yùn)動及腫瘤血管形成等過程[15-16]。近年來發(fā)現(xiàn)，上皮-間質(zhì)轉(zhuǎn)化的異常激活存在于多種病理性過程中，包括上皮來源的腫瘤的纖維化和轉(zhuǎn)移性擴(kuò)散[17]。在腫瘤中，上皮-間質(zhì)轉(zhuǎn)化使得上皮細(xì)胞從原發(fā)腫瘤脫落下來，侵襲到周圍基質(zhì)中，因此，腫瘤性的上皮-間質(zhì)轉(zhuǎn)化是轉(zhuǎn)移進(jìn)展最初階段的重要步驟之一[18]。在侵襲性腫瘤中，有多種信號通路和EMT誘導(dǎo)基因的異常激活，包括TGF-α、Wnt/β-catenin信號通路、Snail/Slug轉(zhuǎn)錄因子家族、Twist轉(zhuǎn)錄因子家族等[19]。

叉頭框家族蛋白是在進(jìn)化過程中高度保守的轉(zhuǎn)錄因子家族，該轉(zhuǎn)錄因子家族成員共包含了17個亞家族(FOXA-R)，目前在人類發(fā)現(xiàn)至少有40多個成員。FOX蛋白家族成員在多種生物學(xué)進(jìn)程包括代謝、發(fā)育、分化、增殖、凋亡、轉(zhuǎn)移、侵襲和壽命中都起著重要的作用。因此，F(xiàn)OX功能的失調(diào)可改變細(xì)胞的命運(yùn)和促進(jìn)腫瘤的發(fā)生。大量的體內(nèi)實驗證明，F(xiàn)OX家族蛋白在發(fā)育和維持組織穩(wěn)態(tài)中起著重要作用[20]。研究表明，F(xiàn)OXC2在胚胎發(fā)育、機(jī)體代謝調(diào)節(jié)、脈管系統(tǒng)的發(fā)育及腫瘤血管生成中起著重要的作用[21-23]。近年來，越來越多的研究表明，F(xiàn)OXC2 在腫瘤發(fā)生及轉(zhuǎn)移過程中起不可忽略的作用。研究顯示，F(xiàn)OXC2通過直接誘導(dǎo)趨化因子4受體和整合素3，從而促進(jìn)腫瘤血管及淋巴管形成[24]。有研究表明FOXC2過表達(dá)促進(jìn)間葉細(xì)胞的分化，誘導(dǎo)基質(zhì)金屬蛋白酶-2(MMP2)和基質(zhì)金屬蛋白酶-9(MMP9)的表達(dá)[25]。有研究顯示P120-catenin 是腫瘤上皮細(xì)胞中穩(wěn)定E-cadherin 的一種調(diào)節(jié)蛋白,FOXC2 能下調(diào)P120-catenin 表達(dá)，從而間接抑制上皮標(biāo)志物E-cadherin 表達(dá)， 導(dǎo)致腫瘤發(fā)生上皮-間質(zhì)轉(zhuǎn)化[26]。 在乳腺癌中，F(xiàn)OXC2的表達(dá)與上皮-間質(zhì)轉(zhuǎn)化和干細(xì)胞特性相關(guān)，干擾FOXC2表達(dá)能抑制細(xì)胞的間質(zhì)表型和相關(guān)的侵襲行為及干細(xì)胞特性，而FOXC2過表達(dá)又能誘導(dǎo)腫瘤干細(xì)胞特性及乳腺癌細(xì)胞的轉(zhuǎn)移[27]。本研究的實驗結(jié)果提示，F(xiàn)OXC2過表達(dá)促使結(jié)直腸癌SW480細(xì)胞發(fā)生顯著的上皮-間質(zhì)轉(zhuǎn)化形態(tài)學(xué)改變。同時，F(xiàn)OXC2過表達(dá)后，SW480細(xì)胞中上皮細(xì)胞標(biāo)志物E-cadherin表達(dá)下調(diào)，而間質(zhì)標(biāo)志物Vmentin、N-cadherin表達(dá)上調(diào)。此外，F(xiàn)OXC2過表達(dá)后，SW480細(xì)胞的侵襲遷移能力增強(qiáng)。這些研究結(jié)果表明，F(xiàn)OXC2在結(jié)直腸癌中的過表達(dá)能誘導(dǎo)上皮-間質(zhì)轉(zhuǎn)化發(fā)生，并增強(qiáng)結(jié)直腸癌細(xì)胞的侵襲遷移能力。

綜上所述，本研究提示FOXC2過表達(dá)可能參與結(jié)直腸癌侵襲轉(zhuǎn)移的早期步驟，更為FOXC2作為新的結(jié)直腸癌轉(zhuǎn)移分子標(biāo)志物提供科學(xué)依據(jù)，對于結(jié)直腸癌轉(zhuǎn)移的臨床干預(yù)治療具有重要科學(xué)意義。

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Effect of FOXC2 on epithelial-mesenchymal transition and invasion of colorectal cancer cells*

Cai Juanjuan,Cui Yanmei,DingYanqing,Liao Wenting△

(Department of Pathological,School of Basic Medical Sciences,Southern MedicalUniversity,Guangzhou,Guangdong 510515,China)

[Abstract]ObjectiveTo identify the role of FOXC2 in the invasion and migration of colorectal cancer cells.MethodsStable cell lines expressing FOXC2(SW480/FOXC2) or vector (SW480/pBabe) were established using retroviral infection method.The morphology alterations of SW480 cells were observed using a microscope.Western blot analysis and immunofluorescence staining assays were performed to detect the expression of E-cadherin,Vimentin and N-cadherin.The invasive and migratory abilities of colorectal cancer cells evaluated using Transwell invasion chamber experiment detection.ResultsThe morphology of SW480 cells was significantly changed after overexpression.From the original shape typical of epithelial cells became spindle shaped growth,similar to the morphology of fibroblasts.Western blot analysis and immunofluorescence staining displayed that overexpression of FOXC2 led to significant downregulation of the epithelial marker E-cadherin,but upregulation of the mesenchymal markers Vimentin and N-cadherin.Transwell assay reveals that overexpression of FOXC2 strongly enhanced the migratory and invasive ability of SW480 cells.ConclusionFOXC2 induces epithelial-mesenchymal transition and promotes the invasive ability of colorectal cancer cells.

[Key words]colorectal neoplasms;FOXC2;epithelial-mesenchymal transition;invasion

doi:·論著·10.3969/j.issn.1671-8348.2016.11.002

* 基金項目：國家自然科學(xué)基金資助項目(U1201226，81472710，81172055)；廣州市珠江科技新星專項項目(2012J2200052，2012J2200044)。

作者簡介：蔡娟娟(1986-)，在讀碩士，主要從事腫瘤研究。△通訊作者，E-mail:liaowt2002@gmail.com。

[中圖分類號]R73

[文獻(xiàn)標(biāo)識碼]A

[文章編號]1671-8348(2016)11-1444-04

(收稿日期：2015-10-11修回日期：2015-12-20)