人類真核翻譯延長因子1A2過表達(dá)對胰腺癌SW1990細(xì)胞體外侵襲及體內(nèi)轉(zhuǎn)移的影響

許朝 胡端敏 章永平 諸琦

·論著·

人類真核翻譯延長因子1A2過表達(dá)對胰腺癌SW1990細(xì)胞體外侵襲及體內(nèi)轉(zhuǎn)移的影響

許朝 胡端敏 章永平 諸琦

目的探討人類真核翻譯延長因子1A2(EEF1A2)過表達(dá)對人胰腺癌SW1990細(xì)胞體外侵襲和肺轉(zhuǎn)移潛能的影響。方法通過攜帶EEF1A2的慢病毒感染人胰腺癌SW1990細(xì)胞建立EEF1A2穩(wěn)定過表達(dá)的SW1990/EEF1A2細(xì)胞株及空質(zhì)粒對照的SW1990/GFP細(xì)胞株，并以親本SW1990細(xì)胞作為空白對照。采用實(shí)時(shí)PCR和蛋白質(zhì)印跡法檢測各組細(xì)胞EEF1A2 mRAN和蛋白的表達(dá)。應(yīng)用Transwell小室檢測細(xì)胞的體外侵襲能力。通過裸鼠尾靜脈注射癌細(xì)胞方法構(gòu)建肺轉(zhuǎn)移模型，8周后觀察并計(jì)數(shù)肺表面的轉(zhuǎn)移結(jié)節(jié)。結(jié)果SW1990/EEF1A2細(xì)胞EEF1A2 mRNA相對表達(dá)量為3.252±0.344，顯著高于SW1990/GFP細(xì)胞的1.000±0.060及親本細(xì)胞的0.944±0.041(t值分別為2.255、2.305，P值均<0.01)；EEF1A2蛋白表達(dá)量為0.833±0.050，顯著高于SW1990/GFP細(xì)胞的0.247±0.035及親本細(xì)胞的0.273±0.041(t值分別為0.572、0.559，P值均<0.01)；穿膜細(xì)胞數(shù)為(60±4)個(gè)，顯著多于SW1990/GFP細(xì)胞的(33±4)個(gè)和親本細(xì)胞的(26±3)個(gè)(t值分別為31.33、34.78，P值均<0.01)；裸鼠肺轉(zhuǎn)移結(jié)節(jié)顯著多于SW1990/GFP細(xì)胞及親本細(xì)胞組(5/5比1/5、1/5，P值均<0.05)。結(jié)論EEF1A2過表達(dá)可以明顯增強(qiáng)胰腺癌SW1990細(xì)胞的體外侵襲和肺轉(zhuǎn)移能力。

胰腺腫瘤； 人類真核翻譯延長因子1A2； 慢病毒感染； 腫瘤侵襲； 腫瘤轉(zhuǎn)移

人類真核翻譯延長因子1A2(eukaryotic translation elongation factor l alpha 2，EEF1A2)位于20q13.3，其編碼蛋白通過結(jié)合氨酰tRNA指導(dǎo)核糖體與mRNA密碼子的結(jié)合在蛋白延伸過程中起重要作用，被認(rèn)為是一種管家基因[1]。近來研究發(fā)現(xiàn)，EEF1A2在多種人類腫瘤中表達(dá)異常增高，如乳腺癌、肝癌、肺癌、前列腺癌等[2-5]，被認(rèn)為是一種癌基因。本課題組前期研究發(fā)現(xiàn)，EEF1A2在正常胰腺組織中不表達(dá)，而在胰腺癌組織中高表達(dá)，過表達(dá)的EEF1A2可增強(qiáng)胰腺癌細(xì)胞的增殖能力和裸鼠體內(nèi)移植瘤的生長[6]。本研究進(jìn)一步探討外源性EEF1A2過表達(dá)對細(xì)胞體外侵襲和體內(nèi)轉(zhuǎn)移的影響。

材料與方法

一、穩(wěn)定過表達(dá)EEF1A2的胰腺癌SW1990細(xì)胞株的構(gòu)建

EEF1A2全長基因(約1.4 kb)由上海吉瑪制藥技術(shù)有限公司合成，基因上、下游分別加上NotⅠ和NsiⅠ酶切序列及保護(hù)堿基。應(yīng)用NotⅠ和NsiⅠ雙酶切合成的EEF1A2片段及pGLV5-EF1a-EGFP/Puro質(zhì)粒(上海吉瑪制藥技術(shù)有限公司)，經(jīng)電泳分離、回收、純化，應(yīng)用T4DNA連接酶進(jìn)行連接，然后轉(zhuǎn)化感受態(tài)DH5a，擴(kuò)增，提取質(zhì)粒。重組質(zhì)粒經(jīng)測序鑒定正確后與包膜蛋白質(zhì)粒、核心蛋白/逆轉(zhuǎn)錄酶質(zhì)粒和載體質(zhì)粒應(yīng)用脂質(zhì)體方法共轉(zhuǎn)染293T細(xì)胞，72 h后收集細(xì)胞培養(yǎng)上清，用0.45 μm過濾器過濾，濾液經(jīng)4℃ 20 000 r/min離心2 h，置-80℃冰箱保存?zhèn)溆谩⒙《驹?10～20 μl)用10% FBS的DMEM培養(yǎng)液10倍稀釋4個(gè)梯度分別感染293T細(xì)胞，通過熒光顯微鏡計(jì)數(shù)熒光細(xì)胞，結(jié)合稀釋倍數(shù)計(jì)算病毒滴度，約為1×109TU/ml。按1.2×106細(xì)胞/孔的密度接種于96孔板，混勻后于37℃ 5% CO2培養(yǎng)24 h。將重組慢病毒顆粒用10% FBS的DMEM細(xì)胞培養(yǎng)液稀釋至1×108TU/ml，吸去96孔板中的培養(yǎng)液，每孔加入上述200 μl稀釋的病毒液。同時(shí)將空病毒載體設(shè)為對照組，于37℃、5% CO2條件下培養(yǎng)，24 h后更換為DMEM完全培養(yǎng)液培養(yǎng)，48 h后更換為含嘌呤霉素(終濃度為0.35 μg/ml)的DMEM完全培養(yǎng)液培養(yǎng)，期間每2 d換液。14 d后得到穩(wěn)定過表達(dá)EEF1A2的人胰腺癌SW1990細(xì)胞(SW1990/ EEF1A2細(xì)胞)和空病毒載體穩(wěn)轉(zhuǎn)的對照SW1990細(xì)胞(SW1990/GFP細(xì)胞)。

二、EEF1A2 mRNA表達(dá)檢測

收集各組細(xì)胞，Trizol試劑提取細(xì)胞總RNA。按反轉(zhuǎn)錄試劑盒說明書提供的方法合成cDNA第一鏈；以cDNA為模板，應(yīng)用實(shí)時(shí)熒光定量PCR 檢測試劑盒(日本TaKaRa公司)行實(shí)時(shí)PCR擴(kuò)增，以GAPDH為內(nèi)參。EEF1A2的上游引物5′-ATGCGG-AGGTATTGACAAAAGGAC-3′，下游引物5′-AGCACC-CCAGGCATACTTGAAGG-3′；擴(kuò)增產(chǎn)物90 bp；內(nèi)參GAPDH的上游引物5′-CATGAGAAGTATGACAAC-AGCCT-3′，下游引物5′-AGTCCTTCCACGATA-CCAAAGT-3′，擴(kuò)增產(chǎn)物113 bp。引物由上海生工生物工程有限公司合成。PCR反應(yīng)條件：95℃ 3 min，95℃ 12 s、62℃ 40 s， 40個(gè)循環(huán)。通過儀器自帶軟件獲取Ct值，根據(jù)公式2-△△Ct計(jì)算EEF1A2 mRNA的相對表達(dá)量，△△Ct=SW1990/EEF1A2[CtEEF1A2-CtGAPDH]-SW1990/GFP[CtEEF1A2-CtGAPDH]。

三、EEF1A2蛋白表達(dá)檢測

提取各組細(xì)胞總蛋白，應(yīng)用BCA法測定蛋白濃度。取50 μg蛋白常規(guī)行蛋白質(zhì)印跡法檢測EEF1A2蛋白的表達(dá)。兔抗人EEF1A2多抗(美國Abcam公司) 1∶1000 稀釋，內(nèi)參GAPDH單抗(上海康成公司)1∶5000 稀釋，HRP標(biāo)記二抗1∶5000 稀釋。應(yīng)用ECL發(fā)光，顯影，Bio-Rad公司數(shù)碼成像分析系統(tǒng)軟件進(jìn)行圖片掃描，以目的條帶與內(nèi)參條帶的灰度比值表示目的蛋白的相對表達(dá)量。

四、Transwell小室侵襲實(shí)驗(yàn)

胰酶消化并收集各組細(xì)胞，用無血清DMEM培養(yǎng)液調(diào)整細(xì)胞密度為1×106個(gè)/ml，取100 μl加入 Matrigel Transwell小室(Chemicon公司)的上室，下室加入含10%胎牛血清的DMEM培養(yǎng)液500 μl，小室放置于24孔板中，常規(guī)培養(yǎng)24 h。棄上室液體，用棉簽輕輕拭去濾膜上表面未穿透的細(xì)胞，經(jīng)甲醇固定15 min，常規(guī)結(jié)晶紫染色，倒置顯微鏡下選擇10個(gè)高倍鏡視野(×200)，計(jì)數(shù)濾膜下表面的穿膜細(xì)胞數(shù)。每組設(shè)3個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次。

五、裸鼠肺轉(zhuǎn)移模型實(shí)驗(yàn)

4～6周齡BALB/C裸鼠購自中國科學(xué)院上海實(shí)驗(yàn)動物中心，飼養(yǎng)于SPF條件下。8周齡時(shí)，將裸鼠按數(shù)字表法隨機(jī)分為2組，每組5只，每只裸鼠經(jīng)尾靜脈注射2×106個(gè)SW1990/EEF1A2細(xì)胞或SW1990/GFP細(xì)胞。8周后脫頸處死裸鼠，分離肺，觀察計(jì)數(shù)肺表面的轉(zhuǎn)移結(jié)節(jié)。

六、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、穩(wěn)定過表達(dá)EEF1A2的胰腺癌SW1990細(xì)胞株的建立

SW1990/EEF1A2、SW1990/GFP和親本SW1990細(xì)胞的EEF1A2 mRNA相對表達(dá)量分別為3.252±0.344、1.000±0.060和0.944±0.041；EEF1A2 蛋白相對表達(dá)量分別為0.833±0.050、0.247±0.035和0.273±0.041。SW1990/EEF1A2細(xì)胞的表達(dá)顯著高于SW1990/GFP細(xì)胞(t值分別為2.255、0.572，P<0.01)，也顯著高于親本SW1990細(xì)胞(t值分別為2.305、0.559，P<0.01，圖1)。

圖1SW1990/GFP、SW1990/EEF1A2細(xì)胞EEF1A2蛋白的表達(dá)

二、EEF1A2過表達(dá)對SW1990細(xì)胞體外侵襲力的影響

SW1990/EEF1A2、SW1990/GFP和SW1990的穿膜細(xì)胞數(shù)分別為(60±4)、(33±4)和(26±3)個(gè)，SW1990/EEF1A2的穿膜細(xì)胞數(shù)顯著高于SW1990/GFP細(xì)胞(t=31.33，P<0.01)，也顯著高于親本SW1990細(xì)胞(t=34.78，P<0.01，圖2)。

圖2SW1990/EEF1A2(a)、SW1990/GFP(b)的穿膜細(xì)胞(結(jié)晶紫染色 ×200)

三、EEF1A2過表達(dá)對SW1990細(xì)胞肺轉(zhuǎn)移的影響

SW1990/GFP和SW1990細(xì)胞組裸鼠中各有1只可見肺表面轉(zhuǎn)移結(jié)節(jié)(1/5)，而SW1990/EEF1A2組裸鼠均見肺表面轉(zhuǎn)移結(jié)節(jié)(5/5)，兩者間差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。

圖3 裸鼠肺表面的轉(zhuǎn)移結(jié)節(jié)

討 論

惡性腫瘤組織EEF1A2的異常表達(dá)不僅可導(dǎo)致腫瘤細(xì)胞增殖及凋亡的失控，在腫瘤侵襲、轉(zhuǎn)移過程中也發(fā)揮重要作用。Edmonds等[7]報(bào)道，EEF1A2在具有轉(zhuǎn)移潛能的大鼠乳腺癌細(xì)胞中表達(dá)水平明顯高于無轉(zhuǎn)移能力的細(xì)胞，過表達(dá)EEF1A2的乳腺癌BT549細(xì)胞可明顯增強(qiáng)腫瘤細(xì)胞的遷移和侵襲能力；相反，沉默乳腺癌HCC1937細(xì)胞EEF1A2表達(dá)則可抑制細(xì)胞的侵襲能力[8-9]。本課題組前期研究也發(fā)現(xiàn)，利用特異性siRNA靶向沉默EEF1A2可抑制胰腺癌細(xì)胞BxPC3體外遷移和侵襲力[10]。

為進(jìn)一步研究EEF1A2對胰腺癌細(xì)胞體內(nèi)轉(zhuǎn)移潛能的影響，我們構(gòu)建了攜帶EEF1A2基因的慢病毒表達(dá)載體，并成功篩選出穩(wěn)定過表達(dá)外源EEF1A2的SW1990細(xì)胞株。結(jié)果顯示EEF1A2過表達(dá)可增強(qiáng)SW1990細(xì)胞的體外侵襲能力及體內(nèi)的肺表面轉(zhuǎn)移。但其分子機(jī)制目前仍不清楚。Amiri等[8]的研究顯示，EEF1A2可激活A(yù)kt信號通路從而促進(jìn)瘤細(xì)胞骨架重構(gòu)和侵襲轉(zhuǎn)移。具體機(jī)制尚有待進(jìn)一步探討。

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(本文編輯：屠振興)

Effectsofhomosapienseukaryotictranslationelongationfactor1alpha2overexpressiononinvitroinvasionandinvivometastasisofpancreatic

cancerSW1990cellsXUChao,HUDuan-min,ZHANGYong-ping,ZHUQi.

DepartmentofGastroenterology,RuijinHospital,ShanghaiJiaotongUniversitySchoolofMedicine,Shanghai200025,China

ZHUQi,Email:zhuqi@medmail.com.cn

ObjectiveTo investigate the effects of over-expression of homo sapiens eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) on in vitro invasion and lung metastasis of human pancreatic cancer SW1990 cells.MethodsLetivirus-mediated delivery of EEF1A2 was used to enhance the expression of EEF1A2 gene in human pancreatic cancer SW1990 cells, the SW1990 cells stably over-expressing EEF1A2 protein (SW1990/EEF1A2 cells) were obtained, and the parent SW1990 cells and SW1990/GFP cells were used as the control, and the expressions of EEF1A2 mRNA and protein were determined by Real-time PCR and Western blotting. The invasion ability of cells was determined by Transwell assay. The lung metastasis model was established by injection of SW1990 cells into the tail vein. Whole lung tissues were harvested, and visible nodules on lung surface were counted macroscopically 8 weeks later.ResultsThe EEF1A2 mRNA expression of SW1990/EEF1A2 was 3.252±0.344, which was significantly higher than those in SW1990/GFP cells (1.000±0.060) and SW1990 cells (0.944±0.041,t=2.255, 2.305,P<0.01); the EEF1A2 protein expression was 0.833±0.050, which was significantly higher than those in SW1990/GFP cells (0.247±0.035) and SW1990 cells (0.273±0.041,t=0.572, 0.559,P<0.01). The ability of invasion of SW1990/EEF1A2 cells was (60±4) cells, which was significantly higher than (33±4) cells in SW1990/GFP

group and (26±3) cells in SW1990 group (t=31.33, 34.78,P<0.01). Furthermore, SW1990/EEF1A2 cells had a much higher incidence of lung metastasis in nude mice than SW1990/GFP cells and SW1990 cells in vivo (100%vs. 20%, 20%,P<0.05).ConclusionsEEF1A2 over-expression can obviously increase the in vitro invasion and lung metastasis of pancreatic cancer SW1990 cells.

Pancreatic neoplasms; Homo sapiens eukaryotic translation elongation factor 1 alpha 2； Lentivirus infections; Neoplasm invasiveness; Neoplasm metastasis

10.3760/cma.j.issn.1674-1935.2013.01.006

200025 上海，上海交通大學(xué)醫(yī)學(xué)院附屬瑞金醫(yī)院消化內(nèi)科

諸琦，Email: zhuqi@medmail.com.cn

2012-09-11)