過表達(dá)HIF-2α對(duì)肝癌細(xì)胞運(yùn)動(dòng)和侵襲的影響

孫海香， 楊柳曉

復(fù)旦大學(xué)附屬中山醫(yī)院肝癌研究所， 上?！?00032

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·短篇論著·

過表達(dá)HIF-2α對(duì)肝癌細(xì)胞運(yùn)動(dòng)和侵襲的影響

孫海香， 楊柳曉*

復(fù)旦大學(xué)附屬中山醫(yī)院肝癌研究所， 上海200032

[摘要]目的： 探討缺氧誘導(dǎo)因子(hypoxia inducible factor-2α, HIF-2α)對(duì)肝癌細(xì)胞運(yùn)動(dòng)和侵襲的影響。方法： 構(gòu)建 HIF-2α過表達(dá)載體，轉(zhuǎn)染肝癌細(xì)胞，構(gòu)建過表達(dá)穩(wěn)定表達(dá)細(xì)胞株。采用實(shí)時(shí)熒光定量多聚酶聯(lián)反應(yīng)(quantitative real-time polymerase chain reaction, qRT-PCR)和蛋白質(zhì)印跡法檢測HIF-2α表達(dá)水平，通過Transwell運(yùn)動(dòng)實(shí)驗(yàn)、劃痕實(shí)驗(yàn)體外分析過表達(dá)HIF-2α對(duì)肝癌細(xì)胞運(yùn)動(dòng)的影響；將過表達(dá)HIF-2α細(xì)胞原位接種裸鼠，觀察小鼠肺臟的轉(zhuǎn)移情況。采用蛋白質(zhì)印跡法檢測EMT相關(guān)指標(biāo)分析HIF-2α促進(jìn)肝癌細(xì)胞運(yùn)動(dòng)、侵襲能力的作用機(jī)制。結(jié)果： 將過表達(dá)載體轉(zhuǎn)染肝癌細(xì)胞后，HIF-2α mRNA和蛋白表達(dá)水平均升高(P<0.05)。Transwell運(yùn)動(dòng)實(shí)驗(yàn)發(fā)現(xiàn)，高表達(dá)HIF-2α后，穿膜細(xì)胞數(shù)目明顯增加(P<0.01)；劃痕實(shí)驗(yàn)結(jié)果也表明高表達(dá)HIF-2α的細(xì)胞運(yùn)動(dòng)能力增強(qiáng)。體內(nèi)侵襲實(shí)驗(yàn)發(fā)現(xiàn)高表達(dá)HIF-2α組小鼠肺轉(zhuǎn)移灶大小增加。蛋白質(zhì)印跡結(jié)果顯示HIF-2α可促進(jìn)間質(zhì)細(xì)胞相關(guān)標(biāo)志物蛋白表達(dá)增加。結(jié)論： 過表達(dá)HIF-2α可能通過促進(jìn)肝癌細(xì)胞的EMT轉(zhuǎn)化提高肝癌細(xì)胞的運(yùn)動(dòng)、侵襲能力。

[關(guān)鍵詞]HIF-2α；肝癌細(xì)胞；運(yùn)動(dòng)；侵襲

缺氧誘導(dǎo)因子2α(hypoxia inducible factors, HIF-2α)是HIFs家族的重要成員，可促進(jìn)c-Myc的轉(zhuǎn)錄活性[1-2]，與VEGF增強(qiáng)子結(jié)合[3]；HIF-2α特異調(diào)節(jié)Oct-4表達(dá)，對(duì)腫瘤干細(xì)胞“干”性維持發(fā)揮關(guān)鍵作用[4]；HIF-2α有血管內(nèi)皮細(xì)胞生長因子受體2(VEGFR-2/Flk-1)、基質(zhì)金屬蛋白酶9(MMP-9)、模型金屬蛋白酶(MT1-MMP)[5-6]等特異的靶基因。

HIF-2α在肝癌組織中廣泛表達(dá)[3]，表達(dá)水平與VEGF相關(guān)，其高表達(dá)與肝癌的惡性程度正相關(guān)，高表達(dá)HIF-2α的患者預(yù)后較差[7]。干擾HIF-2α表達(dá)可抑制轉(zhuǎn)化生長因子α(transforming growth factor alpha, TGF-α)、cyclin D1表達(dá)，提高阿霉素(doxorubicin)的化療效果[8]。但也有研究顯示干擾HIF-2α顯著上調(diào)Bcl-2/腺病毒 E1B相互作用蛋白3(Bcl-2/adenovirus E1B 19kDa-interacting protein 3，BNIP3)的表達(dá)，促進(jìn)自噬小體形成，導(dǎo)致肝癌迅速生長[9]。因此，本研究利用過表達(dá)質(zhì)粒提高肝癌細(xì)胞HIF-2α的表達(dá)，觀察細(xì)胞運(yùn)動(dòng)侵襲能力的變化，為進(jìn)一步探討HIF-2α在肝癌中的確切作用奠定基礎(chǔ)。

1材料與方法

1.1主要材料及試劑肝癌細(xì)胞LM3細(xì)胞株由復(fù)旦大學(xué)附屬中山醫(yī)院肝癌研究所提供，pcDNA3-HIF-2α質(zhì)粒購自上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司，小鼠購自上海靈暢實(shí)驗(yàn)動(dòng)物有限公司。RNA抽提TRIzol試劑(美國Invitrogen公司)；熒光實(shí)時(shí)定量PCR(Quantitative real-time polymerase chain reaction)購買自日本TaKaRa公司；HIF-2α及GAPDH引物(上海生工生物工程有限公司)；細(xì)胞裂解液、蛋白濃度測定試劑盒(江蘇碧云天公司)；HIF-2α及GAPDH、 E-cadherin、 N-cadherin、 Fibronectin、Vimentin抗體(英國Abcam公司)。

1.2RT-PCR檢測HIF-2α基因表達(dá)RNA抽提、cDNA合成等均按照試劑盒說明書進(jìn)行操作； PCR反應(yīng)條件如下：94℃預(yù)變性5 min后，開始PCR循環(huán)：95℃變性15 s，60℃退火30 s，72℃延伸30 s，共30個(gè)循環(huán)。

1.3蛋白質(zhì)印跡法檢測蛋白表達(dá)蛋白抽提先用RIPA，考馬斯亮藍(lán)法(Bradford法)檢測裂解液中蛋白濃度，操作步驟參照說明書。

1.4細(xì)胞劃痕實(shí)驗(yàn)將細(xì)胞按3×104/孔密度接種于24孔板中，設(shè)實(shí)驗(yàn)組和對(duì)照組；待細(xì)胞長至70%～80%匯合度時(shí)，在培養(yǎng)板底部劃“十”字形劃痕；倒置顯微鏡下觀察劃痕后48 h細(xì)胞的遷移情況。

1.5運(yùn)動(dòng)實(shí)驗(yàn)收獲實(shí)驗(yàn)組和對(duì)照組的處于生長對(duì)數(shù)期細(xì)胞，DMEM稀釋液成1×106/mL單細(xì)胞懸液，取100 μL加入上室，下室加入300 μL DMEM培養(yǎng)液，含10%胎牛血清；72 h后用4%多聚甲醛固定10 min；Giemsa染液染色30 min；顯微鏡下計(jì)數(shù)并拍照。

1.6原位瘤種植腫瘤細(xì)胞在裸鼠肝臟原位注射成瘤，6周后處死小鼠，測定腫瘤體積，觀察腹腔等部位有無轉(zhuǎn)移灶。取肺臟標(biāo)本，10%中性甲醛固定，連續(xù)切片，H-E染色，鏡下觀察肺轉(zhuǎn)移灶。

2結(jié)果

2.1轉(zhuǎn)染后HIF-2α mRNA和蛋白表達(dá)水平RT-PCR結(jié)果(圖1)表明，轉(zhuǎn)染質(zhì)粒后，HIF-2α的表達(dá)水平顯著上升(P<0.05)；蛋白質(zhì)印跡檢測結(jié)果也表明實(shí)驗(yàn)組HIF-2α蛋白水平明顯上調(diào)。

圖1　HIF-2α的mRNA和蛋白水平檢測

2.2HIF-2α對(duì)肝癌細(xì)胞運(yùn)動(dòng)能力的影響Transwell小室結(jié)果(圖2)表明：與對(duì)照組細(xì)胞相比，實(shí)驗(yàn)組細(xì)胞穿膜數(shù)顯著增加(P<0.01)，提示HIF-2α可顯著增加細(xì)胞的運(yùn)動(dòng)能力；劃痕實(shí)驗(yàn)結(jié)果(圖3)也表明，實(shí)驗(yàn)組細(xì)胞運(yùn)動(dòng)能力明顯高于對(duì)照組。

2.3HIF--2α對(duì)肝癌細(xì)胞侵襲能力的影響結(jié)果(圖4)表明：對(duì)照組和實(shí)驗(yàn)組均有肺臟轉(zhuǎn)移，但實(shí)驗(yàn)組肺轉(zhuǎn)移灶明顯大于對(duì)照組，表明HIF-2α可提高腫瘤細(xì)胞的轉(zhuǎn)移能力。

圖2　兩組細(xì)胞的Transwell實(shí)驗(yàn)結(jié)果

2.4HIF-2α與EMT指標(biāo)的相關(guān)性結(jié)果(圖5)表明：高表達(dá)HIF-2α可增加N-Cadherin、Fibronectin、Vimentin的表達(dá)，而E-cadherin的表達(dá)量基本沒有變化。該結(jié)果提示HIF-2α可能通過促進(jìn)上皮細(xì)胞向間質(zhì)細(xì)胞的轉(zhuǎn)化，從而提高肝癌細(xì)胞的運(yùn)動(dòng)、侵襲能力。

圖3　兩組細(xì)胞的劃痕實(shí)驗(yàn)結(jié)果

圖4　小鼠肺轉(zhuǎn)移灶H-E 染色

圖5　HIF-2α與EMT相關(guān)指標(biāo)的檢測

3討論

目前，HIFs的研究多集中在HIF-1α，而 HIF-2α在腫瘤中的研究較少，目前僅在腎癌、非小細(xì)胞肺癌、頭頸癌等有部分研究數(shù)據(jù)。免疫組化染色結(jié)果表明63.9%的肝癌組織中HIF-2α表達(dá)量高于對(duì)應(yīng)的癌旁組織，但其分子機(jī)制仍然不明確。研究表明，一方面HIF-2α可以直接促進(jìn)VEGF、EPO等基因轉(zhuǎn)錄，促進(jìn)血管新生，從而促進(jìn)肝癌的轉(zhuǎn)移；另一方面，HIF-2α可以顯著下調(diào)BNIP3的表達(dá)，阻礙自噬小體形成，抑制肝癌迅速生長。

本研究通過改變HIF-2α表達(dá)水平，利用Transwell和劃痕實(shí)驗(yàn)表明提高HIF-2α表達(dá)水平可顯著提高肝癌細(xì)胞的運(yùn)動(dòng)能力，通過裸鼠原位接種肝癌組織，發(fā)現(xiàn)HIF-2α高表達(dá)組的肺轉(zhuǎn)移灶明顯增加。本研究的一系列體內(nèi)、外實(shí)驗(yàn)結(jié)果均表明HIF-2α可顯著提高肝癌細(xì)胞的運(yùn)動(dòng)、侵襲能力。之前有數(shù)據(jù)表明，HIF-2α作為轉(zhuǎn)錄因子可以調(diào)節(jié)Twist、Snail等分子的表達(dá)，其通常與細(xì)胞的EMT轉(zhuǎn)化過程相關(guān)，本研究分析了EMT轉(zhuǎn)化的關(guān)鍵分子標(biāo)志物E-cadherin、N-cadherin、Fibronectin和Vimentin的表達(dá)，結(jié)果表明HIF-2α可以顯著提高間質(zhì)細(xì)胞標(biāo)志物的表達(dá)，提示HIF-2α可能是通過促進(jìn)EMT轉(zhuǎn)化，從而提高肝癌細(xì)胞的運(yùn)動(dòng)和侵襲能力。

參考文獻(xiàn)

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[本文編輯]葉婷， 賈澤軍

[收稿日期]2016-03-10[接受日期]2016-05-02

[基金項(xiàng)目]國家自然科學(xué)基金(81302100, 81502028，81572884d，81372317)，高等學(xué)校博士學(xué)科點(diǎn)專項(xiàng)科研基金(20120071120068)，復(fù)旦大學(xué)附屬中山醫(yī)院優(yōu)秀青年基金 (2015ZSYXQN03). Supported by National Natural Science Foundation of China (81302100, 81502028，81572884d，81372317), the Specialized Research Fund for the Doctoral Program of Higher Education (20120071120068), and Zhongshan Hospital Outstanding Youth Fund of Fudan University (2015ZSYXQN03).

[作者簡介]孫海香，博士，助理研究員. E-mail: sun.haixiang@zs-hospital.sh.cn *通信作者(Corresponding author). Tel: 021-64041990-61074, E-mail: yang.liuxiao@zs-hospital.sh.cn

[中圖分類號(hào)]R 735.7

[文獻(xiàn)標(biāo)志碼]A

Effect of overexpression of HIF-2 alpha on movement and invasion of hepatocellular carcinoma cells

SUN Hai-xiang, YANG Liu-xiao*

Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai200032, China

[Abstract]Objective： To investigate the effects of hypoxia inducible factor 2 alpha (HIF-2α ) on movement and invasion of hepatocellular carcinoma cells (HCC). Methods： The plasmid of over-expression of HIF-2α was constructed and transfected into HCC cells, then the stable expression cell strains were selected. HIF-2α expression levels were detected by quantitative real-time polymerase chain reaction (RT-PCR) and Western Bloting. The effects of HIF-2α over-expression on the movement ability of HCC were analyzed in vitro by Transwell assay and scrach assay. The over-expressed HIF-2α cells were planted in situ in nude mice, and the lung metastasis were observed. we checked the EMT markers by Western Bloting. The mechanism of HIF-2α promoting cancer cell migration and invasion ability was analyzed by Western bloting detecting EMT-related indices. Results： When the plasmid of over-expression was transfected into hepatoma cells, the expression levels of HIF-2α mRNA and protein levels both increased (P<0.05). The Transwell movement experiments showed that after the high expression of HIF-2α , the number of transmembrane cells increased significantly (P< 0.01). The scratch test results also showed that the high expression of HIF-2α enhanced the movement ability of cells. The in vivo invasion experiment showed that the high expression of HIF-2α increased the lung metastasis tumor size in mice significantly. Western bloting results showed that HIF-2α can promote the protein expression of mesenchymal cells-associated markers. Conclusions： HIF-2α might improve the movement and invasion ability of hepatocellular carcinoma cells (HCC) by promoting their epithelial-mesenchymal transition (EMT).

[Key Words]HIF-2α ; hepatocellular carcinoma cells; movement; invasion