范永胂，暢銀娟

（1.浙江醫(yī)院 肛腸科，浙江 杭州 310013；2.浙江大學(xué)附屬婦產(chǎn)科醫(yī)院 婦科，浙江 杭州 310000）

·基 礎(chǔ) 研 究·

EIF5A2在結(jié)直腸癌細(xì)胞中的表達(dá)及在5-Fu化療抵抗中的作用

范永胂1，暢銀娟2

（1.浙江醫(yī)院 肛腸科，浙江 杭州 310013；2.浙江大學(xué)附屬婦產(chǎn)科醫(yī)院 婦科，浙江 杭州 310000）

目的：探討EIF5A2在結(jié)直腸癌細(xì)胞中的表達(dá)及在化療抵抗中的作用。方法：培養(yǎng)對(duì)數(shù)生長(zhǎng)期的人結(jié)直腸癌細(xì)胞系LOVO、SW480及DLD1，用Western blot法測(cè)定三種結(jié)直腸癌細(xì)胞系中EIF5A2的表達(dá)。將人結(jié)直腸癌LOVO細(xì)胞在濃度為0.2 μg/L的5-Fu中培養(yǎng)48 h，用Western blot法測(cè)定EIF5A2的表達(dá)。EIF5A2-siRNA轉(zhuǎn)染LOVO細(xì)胞，將轉(zhuǎn)染后的LOVO細(xì)胞及結(jié)直腸癌LOVO細(xì)胞在0.2 μg/L的5-Fu中培養(yǎng)48 h，用MTT法測(cè)定細(xì)胞生長(zhǎng)抑制率。結(jié)果：EIF5A2在三種結(jié)直腸癌細(xì)胞系中均有表達(dá)，在LOVO細(xì)胞中表達(dá)量最高（P＜0.05）。結(jié)直腸癌LOVO細(xì)胞在5-Fu中培養(yǎng)48 h后 EIF5A2表達(dá)明顯增強(qiáng)（P＜0.05）。EIF-5A2-siRNA轉(zhuǎn)染LOVO細(xì)胞后增加了5-Fu的細(xì)胞抑制率（P＜0.05）。結(jié)論：EIF5A2在人結(jié)直腸癌細(xì)胞中有表達(dá)，并且增強(qiáng)了5-Fu的化療抵抗力。

結(jié)直腸腫瘤；EIF5A2；5-氟尿嘧啶；化療抵抗

結(jié)直腸癌是導(dǎo)致癌癥相關(guān)死亡的最常見病因，每年約有超過1千萬人罹患此病[1]。5-Fu聯(lián)合其他藥物化療可以顯著提高患者生存率[2]，然而化療抵抗給結(jié)直腸癌的治療帶來了巨大挑戰(zhàn)。EIF5A2（eukaryotic translation initiation factor 5A2）是一種真核生物翻譯起始因子，是Guan等[3]從卵巢癌中分離出的一種原癌基因。研究表明EIF5A2蛋白的高表達(dá)與結(jié)直腸癌血管淋巴管浸潤(rùn)、較晚的分期和腫瘤遠(yuǎn)處轉(zhuǎn)移有關(guān)[4]，是預(yù)后不良的獨(dú)立危險(xiǎn)因素[5]。但EIF5A2和化療抵抗的關(guān)系及其機(jī)制目前研究較少，所以在本實(shí)驗(yàn)中，我們將檢測(cè)EIF5A2在三種不同的結(jié)直腸癌細(xì)胞系中的表達(dá)情況，并初步探討EIF5A2在5-Fu化療抵抗中的作用。

1 材料和方法

1.1 材料 人結(jié)直腸癌細(xì)胞系LOVO、SW480、DLD1由本院實(shí)驗(yàn)室保存。RPMI 1640培養(yǎng)基、胎牛血清（FBS）購(gòu)自GIBCO公司?？笶IF5A2抗體、EIF5A2-siRNA購(gòu)自武漢晶賽公司。

1.2 方法

1.2.1 細(xì)胞培養(yǎng)：人結(jié)直腸癌細(xì)胞均在含10%胎牛血清的RPMI1640培養(yǎng)液中，飽和濕度、37 ℃及5% CO2培養(yǎng)箱內(nèi)常規(guī)傳代培養(yǎng)。待細(xì)胞生長(zhǎng)至約70%至90%融合狀態(tài)時(shí)用于實(shí)驗(yàn)。

1.2.2 Western blot法檢測(cè)結(jié)直腸癌細(xì)胞EIF5A2表達(dá)情況：收集對(duì)數(shù)生長(zhǎng)期細(xì)胞LOVO、SW480及DLD1，加入PBS液漂洗，后加入裂解液裂解，在4 ℃、6 700 r/min下離心2 min，收集裂解物總蛋白；將蛋白溶液和5×上樣緩沖液按5∶1混合煮沸5 min，按每孔20 μL上樣進(jìn)行電泳并轉(zhuǎn)印至PVDF膜，經(jīng)5%的脫脂奶粉封閉后，與兔抗EIF5A2（1∶1 000）和內(nèi)參照GAPDH（稀釋1∶10 000）多克隆抗體分別孵育，再與羊抗兔二抗（稀釋1∶5 000）反應(yīng)，增強(qiáng)化學(xué)發(fā)光法顯影，與內(nèi)參照進(jìn)行灰度比較以半定量分析。該實(shí)驗(yàn)重復(fù)3次，分析三種結(jié)直腸癌細(xì)胞系中EIF5A2表達(dá)情況。

1.2.3 5-Fu干預(yù)結(jié)直腸癌細(xì)胞LOVO對(duì)EIF5A2表達(dá)的影響：結(jié)直腸癌細(xì)胞LOVO在0.20 μg/L的5-Fu中培養(yǎng)48 h后，Western blot法（如步驟1.2.2所述）檢測(cè)LOVO細(xì)胞內(nèi)EIF5A2的表達(dá)。

1.2.4 EIF5A2-siRNA轉(zhuǎn)染LOVO細(xì)胞后EIF5A2的表達(dá)：EIF5A2-siRNA轉(zhuǎn)染結(jié)直腸癌細(xì)胞系LOVO，按照Lipo-fectamine 2000說明書進(jìn)行瞬時(shí)轉(zhuǎn)染。轉(zhuǎn)染后24 h Western blot法檢測(cè)細(xì)胞內(nèi)EIF5A2表達(dá)情況。

1.2.5 MTT法檢測(cè)不同細(xì)胞對(duì)5-Fu敏感性：取對(duì)數(shù)生長(zhǎng)期的對(duì)照組LOVO細(xì)胞和EIF5A2-siRNA轉(zhuǎn)染組LOVO細(xì)胞，分別暴露于無5-Fu及濃度為0.2 μg/L的5-Fu中培養(yǎng)48 h，用MTT法自動(dòng)酶標(biāo)讀數(shù)儀測(cè)定每孔細(xì)胞吸光度（A值）。實(shí)驗(yàn)設(shè)空白組、藥物實(shí)驗(yàn)組及對(duì)照組，空白組只加細(xì)胞培養(yǎng)液，對(duì)照組加細(xì)胞和培養(yǎng)液。最后計(jì)算細(xì)胞生長(zhǎng)抑制率（%）=[（對(duì)照組-實(shí)驗(yàn)組）/（對(duì)照組-空白組）]×100%。

1.3 統(tǒng)計(jì)學(xué)處理方法 采用SPSS16.0軟件進(jìn)行分析。數(shù)據(jù)采用表示，組間比較采用Student’s t檢驗(yàn)。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 三種結(jié)直腸癌細(xì)胞系中EIF5A2蛋白表達(dá)情況EIF5A2在直腸癌LOVO、SW480、DLD1細(xì)胞系中均有表達(dá)，在LOVO細(xì)胞系中表達(dá)最高，為（71.49± 0.79）%，SW480為（10.79±0.73）%，DLD1為（22.98± 0.81）%，在LOVO細(xì)胞系中EIF5A2的表達(dá)是SW480中的6.62倍，是DLD1細(xì)胞系中的3.11倍，LOVO細(xì)胞與其他2組細(xì)胞之間差異均有統(tǒng)計(jì)學(xué)意義（P＜0.05）。見圖1。

2.2 5-Fu誘導(dǎo)結(jié)腸癌細(xì)胞LOVO中EIF5A2表達(dá) 將結(jié)直腸癌LOVO細(xì)胞暴露于0.2 μg/L的5-Fu中培養(yǎng)48 h，EIF5A2相對(duì)表達(dá)值為（75.20±0.67）%，與無5-Fu組的（71.49±0.79）%相比表達(dá)增強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。

圖1 LOVO、SW480、DLD1細(xì)胞系中EIF5A2的表達(dá)水平

2.3 EIF5A2-siRNA轉(zhuǎn)染后LOVO細(xì)胞后EIF5A2的表達(dá) EIF5A2-siRNA轉(zhuǎn)染組LOVO細(xì)胞EIF5A2的相對(duì)表達(dá)量為（57.90±0.65）%，與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義（P＜0.05），說明EIF5A2表達(dá)受到抑制。

2.4 EIF5A2-siRNA轉(zhuǎn)染LOVO細(xì)胞提高了對(duì)5-Fu的化療敏感性 在無5-Fu組中，EIF5A2-siRNA組細(xì)胞A值與對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）；0.2 μg/L的5-Fu作用實(shí)驗(yàn)中，和對(duì)照組LOVO細(xì)胞相比，EIF5A2-siRNA組A值明顯低于未轉(zhuǎn)染組（P＜0.05，見表1），其對(duì)應(yīng)的細(xì)胞生長(zhǎng)抑制率分別為72.7%、27.2%，表明沉默EIF5A2可明顯增加5-Fu的化療敏感性，減輕化療抵抗。

表1 5-Fu作用于EIF5A2-siRNA轉(zhuǎn)染組和對(duì)照組LOVO細(xì)胞48 h的A值比較（）

表1 5-Fu作用于EIF5A2-siRNA轉(zhuǎn)染組和對(duì)照組LOVO細(xì)胞48 h的A值比較（）

與對(duì)照組LOVO細(xì)胞比：aP＜0.05

3 討論

EIF5A是真核細(xì)胞內(nèi)包含翻譯后衍生氨基酸8-羥-2，7，10-三氨基葵酸的蛋白質(zhì)，對(duì)于維持細(xì)胞增殖非常重要[6]。EIF5A基因家族的成員包括EIF5A1和EIF5A2，兩者編碼的蛋白質(zhì)氨基酸序列有84%的一致性。EIF5A2位于人類染色3q26區(qū)，由5個(gè)外顯子和4個(gè)內(nèi)含子組成，編碼含有125個(gè)氨基酸的EIF5A2蛋白[7]。在多種腫瘤中3q26染色體常表現(xiàn)擴(kuò)增異常或不穩(wěn)定，該現(xiàn)象暗示著染色體3q26區(qū)存在著腫瘤形成與轉(zhuǎn)移基因[8]。多個(gè)研究表明EIF5A2的過表達(dá)和肝癌[9]、結(jié)直腸癌[4]、卵巢癌[10]及肺癌[11]的預(yù)后呈負(fù)相關(guān)。

本研究表明EIF5A2在結(jié)直腸癌LOVO、SW480、DLD1細(xì)胞系中均有表達(dá)，LOVO中最高，DLD1、SW480次之，將結(jié)直腸癌細(xì)胞LOVO暴露于0.2 μg/L 5-Fu中48 h后EIF5A2的表達(dá)增強(qiáng)。同時(shí)我們將LOVO細(xì)胞EIF5A2表達(dá)沉默后作用于相同濃度5-Fu中，結(jié)果5-Fu對(duì)結(jié)直腸癌細(xì)胞的抑制率增加，5-Fu的化療抵抗性得到明顯改善，由此可以推斷EIF5A2可能是一種化療抵抗基因。

EIF5A2表達(dá)雖和化療抵抗相關(guān)，但其機(jī)制仍然不明。最近研究表明，EIF5A2可通過C-MYC上調(diào)轉(zhuǎn)移相關(guān)蛋白1誘導(dǎo)上皮間質(zhì)轉(zhuǎn)化（epithelial-mesenchymal transition，EMT）并增加結(jié)直腸癌細(xì)胞的侵襲轉(zhuǎn)移能力[4]。而許多證據(jù)顯示在乳腺癌[12]、卵巢癌[13]、胰腺癌[14]、肝癌[15]中化療抵抗和EMT都有聯(lián)系。因此，EIF5A2誘導(dǎo)EMT可能是結(jié)直腸癌細(xì)胞化療抵抗的途徑之一。所以，繼續(xù)研究這一課題將對(duì)結(jié)直腸癌的治療提供理論依據(jù)。

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（本文編輯：丁敏嬌）

The expression of EIF5A2 in colorectal cancer cells and its role in 5-Fu chemoresistance

FAN Yongshen1,CHANG Yinjuan2. 1.Department of Anorectal, Zhejiang Hospital, Hangzhou, 310013; 2.Department of Gynecology, Women’s Hospital School of Medicine Zhejiang University, Hangzhou, 310000

Objective: To research EIF-5A2 overexpression in colorectal cancer and its effect on chemotherapy. Methods: EIF5A2 expression in colorectal carcinoma (CRC) line (LOVO, SW48 and DLD1) was measured by Western-blot. LOVO cells were cultured in 5-Fu for 48 hours, and EIF5A2 expression was measured by Western-blot. LOVO cells were treated with EIF5A2-siRNA and exposed to 0.2 μg/L 5-Fu for 48 hours. Cell toxicity was assayed by MTT. Results: EIF5A2 was expressed in all three different CRC and was highest in LOVO cells (P＜0.05). 5-Fu treatment led to elevated EIF5A2 expression (P＜0.05). The transfection of EIF5A2-siRNA could improve the inhibit rate of LOVO to 5-Fu (P＜0.05). Conclusion: EIF5A2 is expressed in CRC lines and it can enhance the chemoresistance of 5-Fu.

colorectal neoplasm; EIF5A2; 5-Fu; chemoresistance

R735.3

B

10.3969/j.issn.2095-9400.2015.07.011

2014-08-07

范永胂（1980-），男，浙江杭州人，主治醫(yī)師，碩士。