吳海霞，錢程，劉春剛，向俊宇，葉迪，張振芳，張獻(xiàn)全

1 重慶醫(yī)科大學(xué) 附屬第二醫(yī)院腫瘤科，重慶 400010

2 第三軍醫(yī)大學(xué) 西南醫(yī)院 生物治療中心，重慶 400038

結(jié)直腸癌是全球范圍內(nèi)常見的惡性消化道腫瘤之一，其發(fā)病率和死亡率還在逐年上升[1]。目前結(jié)直腸癌的治療措施主要為手術(shù)切除，但由于結(jié)直腸癌細(xì)胞具有較高的侵襲和轉(zhuǎn)移的生物學(xué)特性，故影響結(jié)直腸癌患者治療效果的主要因素為遠(yuǎn)處轉(zhuǎn)移，以肝轉(zhuǎn)移最為常見[2-3]。因此找尋新的腫瘤標(biāo)志物及治療靶標(biāo)對(duì)早期診斷和及時(shí)制止其侵襲轉(zhuǎn)移，對(duì)患者的預(yù)后具有至關(guān)重要的意義。

上皮-間質(zhì)轉(zhuǎn)化 (Epithelial-mesenchymal transition EMT) 是一種常見的病理生理學(xué)現(xiàn)象，是指上皮樣細(xì)胞獲得間質(zhì)細(xì)胞特性的過程，發(fā)生EMT時(shí)，細(xì)胞結(jié)構(gòu)改變、極性消失、細(xì)胞間連接減少而運(yùn)動(dòng)能力則增強(qiáng)，表現(xiàn)出類似間質(zhì)細(xì)胞的表型和遷移能力，如上皮樣標(biāo)志物 E-cadherin表達(dá)降低，而間質(zhì)樣標(biāo)志如Vimentin、Fibronectin則增高[4-8]，近年來EMT被認(rèn)為是腫瘤轉(zhuǎn)移的起始環(huán)節(jié)，成為腫瘤轉(zhuǎn)移研究的熱點(diǎn)[9-11]，因此對(duì)EMT的研究有助于增進(jìn)對(duì)腸癌轉(zhuǎn)移的認(rèn)識(shí)。

叉頭框G1 (Forkhead box G1，F(xiàn)OXG1) 基因，又名腦因子 1基因，是叉頭框 (Forkhead box，F(xiàn)OX) 基因家族里的重要一員，位于14q12，其編碼的轉(zhuǎn)錄因子常特異表達(dá)于人類腦組織，常與神經(jīng)系統(tǒng)的發(fā)育性病變有關(guān)[12-17]，有文獻(xiàn)報(bào)道FOXG1在卵巢癌、肝癌中高表達(dá)，并可作為不良預(yù)后的分子指標(biāo)[18-19]，即 FOXG1通過抑制p21WAF1/CIP1轉(zhuǎn)錄，進(jìn)而增強(qiáng)TGF-b介導(dǎo)的促腫瘤增殖能力抗增殖反應(yīng)，但 FOXG1在結(jié)直腸癌中尚無報(bào)道，且 FOXG1是否能促進(jìn)腫瘤的侵襲轉(zhuǎn)移也未見報(bào)道。本研究首次分析了 FOXG1在結(jié)直腸癌侵襲轉(zhuǎn)移中的作用及機(jī)制。

1 材料與方法

1.1 材料

各類腸癌細(xì)胞由本實(shí)驗(yàn)室保存，DMEM培養(yǎng)基、胎牛血清、胰酶、Lipofectamine 2000轉(zhuǎn)染試劑等均購自 Gibco公司；兔抗人 FOXG1抗體購自 Abcam公司；兔抗人 E-cadherin、Vimentin、鼠抗人Fibronectin抗體購自Cell Signaling 公司；分子克隆和慢病毒包裝等試劑由本實(shí)驗(yàn)室保存；Trizol試劑購自Invitrogen公司；逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)試劑盒購自 TaKaRa公司；所有引物均由金斯瑞公司負(fù)責(zé)合成。

1.2 方法

1.2.1 細(xì)胞培養(yǎng)

人結(jié)腸癌細(xì)胞系 RKO、SW480、SW620、LOVO、DLD-1培養(yǎng)于含10%胎牛血清1640培養(yǎng)基中 (含青鏈霉素) 在37 ℃、5% CO2的培養(yǎng)箱中孵育，當(dāng)細(xì)胞密度達(dá)80%左右進(jìn)行傳代，本實(shí)驗(yàn)采用對(duì)數(shù)生長期中的細(xì)胞進(jìn)行實(shí)驗(yàn)。

1.2.2 Western blotting檢測(cè)

提取總蛋白，制10%膠進(jìn)行SDS-PAGE，轉(zhuǎn)膜2 h，5%脫脂牛奶封閉1 h，一抗孵育4 ℃過夜，一抗比例：GAPDH (1∶1 000)、FOXG1 (1∶700)、E-cadherin (1∶1 000)、Vimentin (1∶1 000)Fibronectin (1∶1 000)，PBST洗1 h每10 min換一次液，二抗室溫孵育1.5 h，PBST再洗1 h，拍照存圖。

1.2.3 質(zhì)粒的構(gòu)建及慢病毒轉(zhuǎn)染RKO細(xì)胞

設(shè)計(jì)并合成 3對(duì) FOXG1的 shRNA片段(shFOXG1)，運(yùn)用DNA重組技術(shù)獲得重組質(zhì)粒，經(jīng)雙酶切技術(shù)及測(cè)序方法鑒定正確后進(jìn)行慢病毒的包裝，包裝后存放于-80 ℃，細(xì)胞分為加入病毒的PLKO.1-shFOXG1實(shí)驗(yàn)組及未經(jīng)處理的陰性對(duì)照組。轉(zhuǎn)染前將細(xì)胞接種于6孔板中，當(dāng)細(xì)胞密度達(dá) 50%左右進(jìn)行細(xì)胞穩(wěn)定轉(zhuǎn)染，轉(zhuǎn)染前 1 h常規(guī)換液，經(jīng)嘌呤霉素篩選約1個(gè)月后可獲得穩(wěn)定的細(xì)胞株。

1.2.4RT-PCR檢測(cè)

以Trizol法常規(guī)提取細(xì)胞中的總RNA，按試劑盒進(jìn)行反轉(zhuǎn)錄反應(yīng)合成cDNA，再以cDNA為模板進(jìn)行實(shí)時(shí)熒光定量 PCR反應(yīng)，反應(yīng)條件為95 ℃ 60 s；95 ℃ 15 s，60 ℃ 60 s共 40 個(gè)循環(huán)，以 GAPDH作為內(nèi)參，Bio-Rad 凝膠成像儀采集圖片，以Folds=2-ΔΔCt來表示實(shí)驗(yàn)組和對(duì)照組中目的基因的表達(dá)倍數(shù)比[20]，各引物序列見表1。

1.2.5 劃痕實(shí)驗(yàn)

將細(xì)胞接種于6孔板中，待細(xì)胞融合率達(dá)到70%?80%時(shí)，使用槍頭比著直尺用力劃線，用PBS輕輕沖洗劃下的細(xì)胞，加無血清培養(yǎng)基繼續(xù)培養(yǎng)，分別在0 h和48 h拍照存圖。

表1 目的基因的引物序列Table 1 The primers of related genes

1.2.6 Transwell檢測(cè)

無血清培養(yǎng)基與Matrigel膠按1∶1配成基質(zhì)膠，按10 μL/室加入包被Transwell小室底部膜的上室面，室溫風(fēng)干，上室面加入含10 000個(gè)細(xì)胞的細(xì)胞懸液，下室面加 900 μL的含 20%的 FBS的培養(yǎng)基，置于37 ℃、5% CO2的培養(yǎng)箱中孵育48 h后用4%多聚甲醛進(jìn)行固定30 min，PBS洗滌2次，用結(jié)晶紫燃料染色30 min，輕擦上室面細(xì)胞，顯微鏡下計(jì)數(shù)穿過上室面得細(xì)胞數(shù)，隨機(jī)取5個(gè)視野拍照取均值。細(xì)胞遷移試驗(yàn)：用未被包被的Transwell小室，轉(zhuǎn)移時(shí)間為24 h，所鋪細(xì)胞量為含5 000個(gè)細(xì)胞的細(xì)胞懸液。其余步驟同侵襲實(shí)驗(yàn)。

1.2.7 統(tǒng)計(jì)學(xué)方法

計(jì)量資料用±s表示，采用SPSS 13.0進(jìn)行兩獨(dú)立樣本的t檢驗(yàn)，P≤0.05有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果與分析

2.1 FOXG1在5種腸癌細(xì)胞系中的表達(dá)及與侵襲能力的相關(guān)性分析

在 RKO、SW480、SW620、LOVO、DLD-1五種腸癌細(xì)胞系中，通過Western blotting檢測(cè)發(fā)現(xiàn)，F(xiàn)OXG1在RKO細(xì)胞中蛋白表達(dá)量最高，而在DLD-1細(xì)胞中表達(dá)最低 (圖1A)，由文獻(xiàn)得知這 5種結(jié)直腸癌細(xì)胞的侵襲力如下：LOVO>SW620>RKO>SW480>DLD-1[21]，通過對(duì)FOXG1蛋白相對(duì)表達(dá)量與相應(yīng)細(xì)胞侵襲能力作相關(guān)性分析發(fā)現(xiàn)，在腸癌細(xì)胞中 FOXG1的表達(dá)量與侵襲能力呈正相關(guān) (圖1B)。

2.2 敲低FOXG1可抑制RKO細(xì)胞侵襲和遷移能力

重組質(zhì)粒構(gòu)建成功，經(jīng)雙酶切技術(shù)及測(cè)序方法鑒定為正確后進(jìn)行慢病毒的包裝、純化及穩(wěn)定轉(zhuǎn)染，經(jīng)篩選后獲得了穩(wěn)定的結(jié)直腸癌細(xì)胞株，再通過3種含shFOXG1沉默序列的慢病毒分別感染RKO細(xì)胞后，Western blotting檢測(cè)發(fā)現(xiàn)，與對(duì)照組相比，shFOXG1-3干擾片段對(duì)FOXG1干擾沉默效果最明顯 (圖 2A)。劃痕實(shí)驗(yàn)結(jié)果表明在RKO細(xì)胞中干擾FOXG1后，其遷移能力顯著變?nèi)?圖2B)。Transwell結(jié)果表明干擾FOXG1后，其侵襲和遷移能力也顯著下降 (圖 2C)，由于shFOXG1-3干擾片段對(duì)FOXG1干擾沉默效果最明顯，故后續(xù)實(shí)驗(yàn)均以shFOXG1-3慢病毒轉(zhuǎn)染細(xì)胞，并命名為干擾組 (Interfered group)，單獨(dú)使用時(shí)簡(jiǎn)寫為shFOXG1。

2.3 過表達(dá)FOXG1促進(jìn)DLD-1細(xì)胞侵襲和遷移能力

在DLD-1細(xì)胞過表達(dá)FOXG1后，與對(duì)照組相比較，F(xiàn)OXG1蛋白顯著升高 (圖3A)。RT-qPCR進(jìn)一步驗(yàn)證過表達(dá)效率 (圖 3B)。Transwell結(jié)果表明，過表達(dá)FOXG1促進(jìn)了DLD-1細(xì)胞的侵襲和遷移能力 (圖3C)。

圖 1 在結(jié)直腸癌細(xì)胞中 FOXG1的表達(dá)與侵襲能力呈正相關(guān)Fig. 1 FOXG1 expression of colorectal cancer cells was positively correlated with their invasion ability. (A)FOXG1 expression in colorectal cancer cell lines were analyzed by Western blotting. (B) The correlation between FOXG1 expression and cell invasive abilities was analyzed (Pearson correlation assy).

圖2 干擾FOXG1抑制RKO細(xì)胞侵襲和遷移能力Fig. 2 Knockdown of FOXG1 weakened invasion and migration abilities of RKO cells. (A) The interference efficiency of FOXG1 protein was detected by Western blotting after three virus-infected RKO cells containing shFOXG1 silencing sequence (P<0.05). (B) Healing ability was tested by scratch test. (C) Transwell assay show invasion and migration abilities of FOXG1 Knockdown RKO cells compared with corresponding control cells. *: P<0.05;**: P<0.01; ***: P<0.001.

2.4 FOXG1對(duì)維持結(jié)腸癌EMT具有重要作用

干擾 FOXG1后細(xì)胞形態(tài)由典型的長梭型變成類圓形或多邊形，生長方式也由較為分散轉(zhuǎn)為較密集的生長 (圖 4A)。干擾 FOXG1，Western blotting檢測(cè)分析發(fā)現(xiàn)上皮樣標(biāo)志物E-cadherin表達(dá)在mRNA和蛋白水平都增加，而間質(zhì)類標(biāo)志物Vimentin、Fibronectin表達(dá)降低 (圖4B)。而過表達(dá)FOXG1則相反 (圖4C)。

圖3 過表達(dá)FOXG1促進(jìn)DLD-1細(xì)胞侵襲和遷移能力Fig. 3 Overexpression of FOXG1 promotes invasion and migration abilities of DLD-1 cells. (A) The overexpression efficiency of FOXG1 protein was detected by Western blotting. (B) The overexpression efficiency of FOXG1 mRNA was detected by RT-qPCR. (C) Transwell assay show invasion and migration abilities of FOXG1 overexpression DLD-1 cells compared with corresponding control cells. **: P<0.01; **: P<0.001.

圖4 FOXG1對(duì)維持EMT具有重要作用Fig. 4 FOXG1 plays an important role in maintaining EMT. (A) Morphological changes in RKO cells after stable FOXG1 expression deletion. (B) Expression changes of EMT markers were analyzed by RT-qPCR and Western blotting in RKO cells after FOXG1 knockdown. (C) Expression changes of EMT markers were analyzed by RT-qPCR and Western blotting in DLD-1 cells after overexpression of FOXG1. *: P<0.05; **: P<0.01; ***: P<0.001.

3 討論

研究表明FOX基因家族與腫瘤EMT相關(guān)，如FOXQ1、FOXC1、FOXC2、FOXM1能夠降低E-cadherin表達(dá)，促進(jìn)腫瘤的轉(zhuǎn)移并發(fā)生EMT[22-23]。FOXG1是 FOX基因家族里的重要一員，位于14q12，其編碼的轉(zhuǎn)錄因子常特異表達(dá)于人類腦組織，常與神經(jīng)系統(tǒng)的發(fā)育性病變有關(guān)[12]，它涉及神經(jīng)細(xì)胞的分化、增殖、轉(zhuǎn)移和凋亡，對(duì)端腦的發(fā)育有重要作用[24]，其異常表達(dá)常致 FOXG1的相關(guān)疾病[25-26]，如發(fā)育遲緩、語言障礙、智力低下等。有研究表明，F(xiàn)OXG1在卵巢癌、肝癌中高表達(dá)并作為促癌基因，通過對(duì)P21的抑制促進(jìn)癌細(xì)胞的增殖[18-19]，這提示我們 FOXG1可能與腫瘤的發(fā)生發(fā)展有關(guān)。而 FOXG1在結(jié)直腸癌中表達(dá)情況尚無報(bào)道，且之前對(duì)于 FOXG1在某些實(shí)體瘤的研究主要局限于增殖方向，F(xiàn)OXG1是否能促進(jìn)腫瘤的其他生物學(xué)功能還不明確，且大量研究表明 EMT與結(jié)直腸癌的侵襲和轉(zhuǎn)移有關(guān)[27]，故本研究在細(xì)胞水平上探索了在直結(jié)腸癌中FOXG1與EMT和轉(zhuǎn)移的關(guān)系?；谏鲜觯殚員CGA數(shù)據(jù)庫得知在結(jié)直腸癌組織中 FOXG1的表達(dá)高于正常組織，同時(shí)本研究檢測(cè)了5種結(jié)直腸癌中 FOXG1的蛋白表達(dá)水平，并作了蛋白相對(duì)表達(dá)量與細(xì)胞侵襲能力相關(guān)性分析 (Pearson相關(guān)性檢驗(yàn))，最終確定干擾和過表達(dá)的細(xì)胞系即對(duì)RKO細(xì)胞進(jìn)行干擾，DLD-1細(xì)胞進(jìn)行過表達(dá)，并檢測(cè)對(duì)結(jié)直腸癌細(xì)胞侵襲轉(zhuǎn)移能力的影響。結(jié)果表明慢病毒干擾載體PLKO.1-shFOXG1成功沉默了FOXG1的表達(dá)后，RKO細(xì)胞的侵襲轉(zhuǎn)移能力下降，干擾FOXG1后RKO細(xì)胞形態(tài)由典型的長梭型變成類圓形或多邊形，細(xì)胞形態(tài)變得飽滿，細(xì)胞間的緊密連接增多生長也變得較為密集，且上皮類標(biāo)志物 E-cadherin表達(dá)增高，而間質(zhì)類標(biāo)志物 Vimentin、Fibronectin表達(dá)降低，過表達(dá)組則相反，這些結(jié)果表明 FOXG1可能是腫瘤細(xì)胞維持間質(zhì)樣表型所必需的，但具體機(jī)制還需進(jìn)一步研究。

綜上所述，本研究證明 FOXG1能促進(jìn)結(jié)直腸癌細(xì)胞侵襲和轉(zhuǎn)移，并促進(jìn)其發(fā)生EMT，有望成為一個(gè)新的治療靶點(diǎn)，但其具體機(jī)制還需進(jìn)一步探究。

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