王雯珺，伍思培，列璞怡，郭敏章，何建行

·論著·

LMO4基因沉默對Snail誘導的非小細胞肺癌A549細胞上皮-間質轉化的影響研究

王雯珺，伍思培，列璞怡，郭敏章，何建行

510120廣東省廣州市，呼吸疾病國家重點實驗室(王雯珺，列璞怡，郭敏章，何建行)；廣州醫(yī)科大學第一附屬醫(yī)院(王雯珺，列璞怡，郭敏章，何建行)；廣州醫(yī)科大學(王雯珺，列璞怡，郭敏章，何建行)；廣東省人民醫(yī)院腫瘤中心(伍思培)；廣東省醫(yī)學科學院(伍思培)

【摘要】目的探討LMO4基因沉默對Snail誘導的非小細胞肺癌A549細胞上皮-間質轉化(EMT)的影響。方法體外培養(yǎng)非小細胞肺癌A549細胞，取對數(shù)生長期細胞進行實驗，根據(jù)轉染慢病毒分為sh-NC組(轉染sh-NC慢病毒)、sh-LMO4組(轉染sh-LMO4慢病毒)、Snail組(轉染Snail慢病毒)和Snail+sh-LMO4組(轉染Snail慢病毒和sh-LMO4慢病毒)。采用實時定量RT-PCR檢測sh-NC組和sh-LMO4組LMO4 mRNA相對表達量，采用Western Bloting法檢測sh-NC組和sh-LMO4組LMO4蛋白相對表達量及4組N-cadherin、Vimentin、E-cadherin蛋白相對表達量，采用Transwell實驗檢測4組細胞侵襲個數(shù)，采用細胞劃痕實驗檢測4組細胞遷移距離。結果sh-LMO4組LMO4 mRNA相對表達量和LMO4蛋白相對表達量均低于sh-NC組(P<0.05)。Snail組侵襲細胞個數(shù)多于sh-NC組，sh-LMO4組侵襲細胞個數(shù)少于sh-NC組，Snail+sh-LMO4組侵襲細胞個數(shù)少于Snail組、多于sh-LMO4組(P<0.05)。培養(yǎng)24 h后Snail組細胞遷移距離長于sh-NC組，sh-LMO4組細胞遷移距離短于sh-NC組，而Snail+sh-LMO4組細胞遷移距離短于Snail組、長于sh-LMO4組(P<0.05)。Snail組N-cadherin和Vimentin蛋白相對表達量高于sh-NC組，E-cadherin蛋白相對表達量低于sh-NC組(P<0.05)；sh-LMO4組N-cadherin和Vimentin蛋白相對表達量低于sh-NC組，E-cadherin蛋白相對表達量高于sh-NC組(P<0.05)；Snail+sh-LMO4組N-cadherin和Vimentin蛋白相對表達量低于Snail組，E-cadherin蛋白相對表達量高于Snail組(P<0.05)；Snail+sh-LMO4組N-cadherin和Vimentin蛋白相對表達量高于sh-LMO4組，E-cadherin蛋白相對表達量低于sh-LMO4組(P<0.05)。結論LMO4基因沉默可逆轉Snail誘導的非小細胞肺癌A549細胞EMT。

【關鍵詞】癌，非小細胞肺；LMO4；Snail；上皮-間質轉化

王雯珺，伍思培，列璞怡，等.LMO4基因沉默對Snail誘導的非小細胞肺癌A549細胞上皮-間質轉化的影響研究[J].實用心腦肺血管病雜志，2016，24(5)：54-58.[www.syxnf.net]

Wang WJ， Wu SP，Lie PY，et al.Impact of LMO4 gene silencing on snail-induced EMT of non-small cell lung cancer A549 cells[J].Practical Journal of Cardiac Cerebral Pneumal and Vascular Disease，2016，24(5)：54-58.

在我國肺癌的發(fā)病率在所有腫瘤疾病中居首位，目前其主要治療方法是以手術治療為主的綜合治療，而導致患者死亡的主要原因是復發(fā)或腫瘤轉移[1]。因此，了解腫瘤轉移過程中分子作用機制有利于尋找有效的治療靶點，為提高患者的臨床預后提供可能。LMO4是最新發(fā)現(xiàn)的核轉錄協(xié)作因子，屬于LIM蛋白家族中的一員，其可作為一個橋連接因子與其他組織特定部位的DNA結合蛋白發(fā)生相互作用，形成多蛋白復合體，進而調節(jié)基因轉錄。有研究指出，LMO4在多種上皮細胞癌組織中呈高表達，包括前列腺癌、乳腺癌、口腔癌、神經(jīng)母細胞瘤等[2-5]。據(jù)報道LMO4在乳腺癌的細胞周期進程中起重要作用，且有研究者認為LMO4可作為乳腺癌的獨立預測因素[3]。Kim等[6]學者認為，LMO4可能通過影響肺癌細胞上皮-間質轉化(EMT)而引起腫瘤轉移，已明確Snail可促進細胞發(fā)生EMT。本研究采用sh-LMO4慢病毒處理被Snail慢病毒轉染的非小細胞肺癌A549細胞，旨在探討LMO4基因沉默對Snail誘導的非小細胞肺癌A549細胞EMT的影響。

1材料與方法

1.1細胞培養(yǎng)人非小細胞肺癌A549細胞株為本實驗室自存，將人非小細胞肺癌A549細胞于含有10%胎牛血清(Hyclone，美國)、100 U/ml青霉素、100 μg/ml鏈霉素(碧云天，杭州)的1640培養(yǎng)基(Gibico，美國)中傳代培養(yǎng)，培養(yǎng)條件：溫度37 ℃，5%二氧化碳。A549細胞經(jīng)過消化傳代，取對數(shù)生長期細胞進行實驗。

1.2實驗分組及慢病毒轉染sh-LMO4慢病毒、sh-NC慢病毒和Snail慢病毒購于吉凱公司，慢病毒轉染前24 h，將處于對數(shù)生長期的目的細胞消化后接種于6孔板中(細胞數(shù)約為5×105)過夜；用含有6 μg/ml Polybrene的新鮮培養(yǎng)基2 ml換液，根據(jù)預實驗摸索的細胞感染復數(shù)(MOI)值，分別加入適量的sh-NC慢病毒(sh-NC組)、sh-LMO4慢病毒(sh-LMO4組)和Snail慢病毒(Snail組)，37 ℃孵育。繼續(xù)培養(yǎng)24 h后換液，轉染3 d后采用含嘌呤霉素(濃度為5.00 μg/ml)的培養(yǎng)基繼續(xù)培養(yǎng)并進行篩選，選取穩(wěn)定細胞株進行后續(xù)實驗。然后在轉染Snail慢病毒的細胞中轉染sh-LMO4慢病毒(Snail+sh-LMO4組)，轉染3 d后用含G418(濃度5.00 μg/ml)的培養(yǎng)基繼續(xù)培養(yǎng)并進行篩選，選取穩(wěn)定細胞株進行后續(xù)實驗。

名詞解釋：

轉染是將外源性基因導入細胞內的一種專門技術，大致可分為物理介導、化學介導和生物介導3類。物理介導方法包括電穿孔法、顯微注射和基因槍；化學介導方法有很多，如經(jīng)典的磷酸鈣共沉淀法、脂質體轉染法和多種陽離子物質介導的技術；生物介導方法包括原始的原生質體轉染和各種病毒介導的轉染技術。

1.3實時定量RT-PCRTrizol法提取sh-NC組和sh-LMO4組細胞總RNA，1%瓊脂糖凝膠電泳，定量后使用TAKARA反轉錄試劑盒(1st Strand cDNA Synthesis Kit)進行反轉錄，行特定引物PCR擴增，引物合成在吉瑪公司進行，具體操作按照說明書執(zhí)行，采用2-ΔΔct法計算LMO4 mRNA的相對表達量。PCR引物如下：LMO4上游引物：5′-GGA CCG CTT TCT GCT CTA TG-3′；LMO4下游引物：5′-AAG CAC CGC TAT TCC CAA AT-3′；GAPDH上游引物：5′-AAT CCC ATC ACC ATC TTC CA-3′，GAPDH下游引物：5′-CCT GCT TCA CCA CCT TCT TG-3′。

1.4Western Bloting法提取4組細胞總蛋白，進行蛋白定量檢測，電泳后轉膜，Western封閉液37 ℃封閉2 h，加入相應的一抗(santa cruz，美國)，抗體稀釋比例分別為ant-LMO4(1∶1 000)、anti-N-cadherin(1∶1 000)、anti-Vimentin(1∶1 000)、anti-E-cadherin(1∶1 000)、anti-GAPDH(1∶500)，4 ℃孵育過夜；然后加入稀釋比例為1∶500的辣根過氧化酶標記的二抗(santa cruz，USA)?；瘜W發(fā)光試劑盒在膠片上顯影，晾干后掃描，測定灰度值，并計算擴增出的目的基因與內參基因灰度值的比值，即以LMO4/GAPDH灰度值的比值表示LMO4蛋白相對表達量、N-cadherin/GAPDH灰度值的比值表示N-cadherin蛋白相對表達量、Vimentin /GAPDH灰度值的比值表示Vimentin 蛋白相對表達量、E-cadherin/GAPDH灰度值的比值表示E-cadherin蛋白相對表達量。

1.5Transwell實驗將4組細胞制備成單細胞懸液，在Transwell上室(Gibico，美國)中鋪好基質膠(BD，美國)，每孔加入無血清培養(yǎng)液200 μl(含細胞5×104個/ml)，下室加入500 μl含10%胎牛血清的RPMI-1640培養(yǎng)液，常規(guī)培養(yǎng)24 h后用棉簽擦去基質膠和上室內的細胞進行染色，使用奧林巴斯倒置顯微鏡拍照(×200)，取若干視野計數(shù)侵襲細胞個數(shù)。

1.6細胞劃痕實驗取4組細胞，消化后以5×106/L密度接種于12孔細胞培養(yǎng)板，用200 μl槍頭在每孔中央劃出一道劃痕，處理24 h后洗去死細胞，采用奧林巴斯顯微鏡拍照記錄細胞生長情況。采用Image Pro Plus 6.0軟件測量每孔多個點劃痕間距，取平均值。

2結果

2.1sh-LMO4慢病毒轉染非小細胞肺癌A549細胞后LMO4表達情況sh-LMO4組LMO4 mRNA相對表達量和LMO4蛋白相對表達量均低于sh-NC組，差異有統(tǒng)計學意義(P<0.05，見表1、圖1)。

注：A采用實時定量RT-PCR，B采用Western Bloting法

圖1sh-NC組和sh-LMO4組LMO4 mRNA和LMO4蛋白表達情況

Figure 1Expression of LMO4 mRNA and LMO4 protein of sh-NC group and sh-LMO4 group

Table 1Comparison of the relative expression quantity of LMO4 mRNA and LMO4 protein between sh-NC group and sh-LMO4 group

組別LMO4mRNA相對表達量LMO4蛋白相對表達量sh-NC組1.10±0.110.97±0.14sh-LMO4組0.28±0.060.38±0.06t值5.513.71P值0.000.02

2.2LMO4基因沉默對Snail誘導的非小細胞肺癌A549細胞侵襲能力的影響Transwell實驗結果顯示，sh-NC組侵襲細胞個數(shù)為(129.66±15.91)個，Snail組為(311.11±15.91)個，sh-LMO4組為(58.00±1.73)個，Snail+sh-LMO4組為(131.66±6.00)個。4組侵襲細胞個數(shù)比較，差異有統(tǒng)計學意義(F=43.99，P=0.00)，其中Snail組侵襲細胞個數(shù)多于sh-NC組，差異有統(tǒng)計學意義(t=6.94，P=0.02)；sh-LMO4組侵襲細胞個數(shù)少于sh-NC組，差異有統(tǒng)計學意義(t=7.76，P=0.00)；Snail+sh-LMO4組侵襲細胞個數(shù)少于Snail組、多于sh-LMO4組，差異有統(tǒng)計學意義(t值分別為18.18、9.58，P<0.05，見圖2)。

圖2　4組細胞侵襲能力

2.3LMO4基因沉默對Snail誘導的非小細胞肺癌A549細胞遷移能力的影響細胞劃痕實驗結果顯示，24 h后sh-NC組細胞遷移距離為(15.17±3.42)μm，Snail組為(63.24±8.71)μm，sh-LMO4組為(5.17±0.92)μm，Snail+sh-LMO4組為(31.23±3.80)μm。4組細胞遷移距離比較，差異有統(tǒng)計學意義(F=28.54，P=0.00)，其中Snail組細胞遷移距離長于sh-NC組，差異有統(tǒng)計學意義(t=4.88，P=0.04)；sh-LMO4組細胞遷移距離短于sh-NC組，差異有統(tǒng)計學意義(t=4.89，P=0.01)；Snail+sh-LMO4組細胞遷移距離短于Snail組、長于sh-LMO4組，差異有統(tǒng)計學意義(t值分別為5.83、7.01，P<0.05，見圖3)。

2.4LMO4基因沉默對Snail誘導的非小細胞肺癌A549細胞N-cadherin、Vimentin、E-cadherin表達的影響4組E-cadherin、N-cadherin、Vimentin蛋白相對表達量比較，差異有統(tǒng)計學意義(P<0.05)。Snail組N-cadherin和Vimentin蛋白相對表達量高于sh-NC組，E-cadherin蛋白相對表達量低于sh-NC組，差異有統(tǒng)計學意義(P<0.05)；sh-LMO4組N-cadherin和Vimentin蛋白相對表達量低于sh-NC組，E-cadherin蛋白相對表達量高于sh-NC組，差異有統(tǒng)計學意義(P<0.05)；Snail+sh-LMO4組N-cadherin和Vimentin蛋白相對表達量低于Snail組，E-cadherin蛋白相對表達量高于Snail組，差異有統(tǒng)計學意義(P<0.05)；Snail+sh-LMO4組N-cadherin和Vimentin蛋白相對表達量高于sh-LMO4組，E-cadherin蛋白相對表達量低于sh-LMO4組，差異有統(tǒng)計學意義(P<0.05，見表2、圖4)。

圖3　4組細胞遷移能力

圖4　4組E-cadherin、N-cadherin、Vimentin蛋白表達情況

Figure 4The expression of E-cadherin protein，N-cadherin protein and Vimentin protein of the four groups

表24組N-cadherin、Vimentin、E-cadherin蛋白相對表達量比較

Table 2Comparison of the relative expression quantity of N-cadherin protein，Vimentin protein and E-cadherin protein of the four groups

組別N-cadherin蛋白相對表達量Vimentin蛋白相對表達量E-cadherin蛋白相對表達量sh-NC組0.22±0.100.17±0.530.67±0.04Snail組0.59±0.06ab0.48±0.05ab0.25±0.04absh-LMO4組0.11±0.10ab0.13±0.01ab0.74±0.03abSnail+sh-LMO4組0.39±0.330.35±0.060.43±0.11F值48.959.4925.59P值0.000.000.00

注：與sh-NC組比較，aP<0.05;與Snail+sh-LMO4組比較，bP<0.05

3討論

肺癌是全球范圍內發(fā)病率和病死率最高的惡性腫瘤，在我國肺癌的發(fā)病率近年來呈逐年上升趨勢，目前手術仍是其主要治療方法。近年來，雖然CT、正電子發(fā)射斷層掃描(PET)-CT等影像學技術迅速發(fā)展，但仍存在漏診和誤診現(xiàn)象，很多肺癌患者就診時已是中晚期，已錯過最佳治療時間。臨床研究表明，晚期腫瘤患者多存在多臟器的侵襲和轉移，如能在基因、蛋白質層面發(fā)現(xiàn)早期診斷、預測腫瘤的標志物，或阻斷侵襲和轉移途徑中的關鍵基因，則對晚期肺癌患者的治療具有重要意義，因此該方面的研究已引起廣大醫(yī)療工作者和科研人員的關注。

LIM蛋白是一個非常重要的轉錄調節(jié)因子，其在決定細胞生長與調亡、胚胎發(fā)育及腫瘤發(fā)生中具有重要作用。LMO蛋白是LIM的亞家族，有LMO1～LMO4 4個家族成員，LMO蛋白主要定位于細胞核，含有可與蛋白質相互作用的LIM鋅結合域，但缺乏DNA結合及催化結構域[7]。LMO家族在正常器官發(fā)育中起關鍵作用，且動物實驗結果表明，缺乏LMO1和LMO3的小鼠出生后會出現(xiàn)不明原因死亡[8]。臨床研究顯示，LMO1的表達與T淋巴細胞白血病、急性淋巴細胞白血病密切相關[9]；LMO2在胚胎造血和血管生成中是必不可少的，在血管內皮細胞中下調LMO2表達可使 E-cadherin表達下調[10]；LMO4作為LMO家族中的一員，含有156個氨基酸的殘基，其基本功能是與其他DNA結合蛋白組成大的蛋白復合體，調節(jié)轉錄因子的活性，從而調節(jié)一系列基因的表達，在胚胎發(fā)育、組織分化及腫瘤形成過程中起重要作用。LMO4可與p53相互作用，但過表達的p53可抑制LMO4[11]。有研究顯示，LMO4可調節(jié)轉化生長因子β(TGF-β)引起的EMT[12]。

在上皮細胞癌中，絕大部分的轉移與EMT的發(fā)生有關，且同時伴隨明顯的細胞形態(tài)學改變、細胞與細胞間及細胞與基質間的黏附性丟失、遷移和侵襲能力增強等。EMT的發(fā)生主要以上皮標志物E-cadherin下降和間質標志物Vimentin上升為生物學特征。研究人員通過在原發(fā)性腫瘤中檢測EMT相關標志物發(fā)現(xiàn)，其表達與腫瘤的轉移及患者預后有關。有研究指出在Snail介導的EMT中LMO4起關鍵作用。Snail屬于含有SNAG結構域的鋅指(zinc-finger)蛋白家族成員，SNAG位于Snail分子的N末端，具有抑制基因轉錄的作用[13]。在胚胎發(fā)育和腫瘤轉移過程中，Snail是誘導EMT的關鍵因子，是連接多種促腫瘤轉移信號通路的中心分子[14]。本實驗旨在探討LMO4基因沉默對Snail誘導的非小細胞肺癌A549細胞EMT的影響。

本研究結果顯示，sh-LMO4組LMO4 mRNA相對表達量和LMO4蛋白相對表達量均低于sh-NC組，提示sh-LMO4慢病毒能抑制非小細胞肺癌A549細胞中LMO4的表達；Snail組侵襲細胞個數(shù)多于sh-NC組、細胞遷移距離長于sh-NC組，sh-LMO4組侵襲細胞個數(shù)少于sh-NC組、細胞遷移距離短于sh-NC組，提示Snail過表達能增強腫瘤細胞的侵襲和遷移能力，進而促進EMT的發(fā)生；LMO4基因沉默能減弱腫瘤細胞的侵襲和遷移能力，進而抑制EMT的發(fā)生。本研究結果亦顯示，Snail+sh-LMO4組侵襲細胞個數(shù)少于Snail組、多于sh-LMO4組，細胞遷移距離短于Snail組、長于sh-LMO4組，提示LMO4基因沉默可抑制過表達的Snail對非小細胞肺癌A549細胞侵襲和遷移能力的增強作用。E-cadherin是上皮細胞標志物，N-cadherin、Vimentin是間質細胞標志物，研究顯示E-cadherin蛋白表達降低及N-cadherin、Vimentin蛋白表達升高提示EMT的發(fā)生。本研究結果顯示，Snail組N-cadherin和Vimentin蛋白相對表達量高于sh-NC組，E-cadherin蛋白相對表達量低于sh-NC組；LMO4組N-cadherin和Vimentin蛋白相對表達量低于sh-NC組，E-cadherin蛋白相對表達量高于sh-NC組；Snail+sh-LMO4組N-cadherin和Vimentin蛋白相對表達量低于Snail組，E-cadherin蛋白相對表達量高于Snail組；Snail+sh-LMO4組N-cadherin和Vimentin蛋白相對表達量高于sh-LMO4組，E-cadherin蛋白相對表達量低于sh-LMO4組。提示Snail過表達能促進EMT的發(fā)生，LMO4基因沉默能抑制EMT的發(fā)生，且LMO4基因沉默可逆轉Snail誘導的A549細胞發(fā)生EMT。

綜上所述，LMO4沉默可逆轉Snail誘導的非小細胞肺癌A549細胞發(fā)生EMT，為后續(xù)研究LMO4對肺癌的侵襲和轉移的分子機制提供了實驗依據(jù)。

作者貢獻：王雯珺進行實驗設計與實施、資料收集整理、撰寫論文、成文并對文章負責；伍思培、列璞怡、郭敏章進行實驗實施、評估、資料收集；何建行進行質量控制。

本文無利益沖突。

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(本文編輯：謝武英)

Impact of LMO4 Gene Silencing on Snail-induced EMT of Non-small Cell Lung Cancer A549 Cells

WANGWen-jun，WUSi-pei，LIEPu-yi，etal.

NationalKeyLaboratoryforRespiratoryDisease，Guangzhou510120，China

【Abstract】ObjectiveTo investigate the impact of LMO4 gene silencing on Snail-induced EMT of non-small cell lung cancer A549 cells.MethodsNon-small cell lung cancer A549 cells were cultured in vitro，and logarithmic growth phase cells were extracted to carry out the experiment.According to the transfection of slow viruses，the cells were divided into A group(transfected with sh-NC slow virus)，B group(transfected with sh-LMO4 slow virus)，C group(transfected with Snail slow virus)and D group(transfected with Snail and sh-LMO4 slow virus).Quantitative Real-Time RT-PCR was used to detect the relative expression quantity of LMO4 mRNA of A group and B group；Western Bloting method was used to detect the relative expression quantity of LMO4 protein of A group and B group，the relative expression quantity of N-cadherin protein，Vimentin protein and E-cadherin protein of the four groups；Transwell test was used to detect the number of cell invasion of the four groups；cell scratch test was used to detect the cell migration distance of the four groups.ResultsThe relative expression quantity of LMO4 mRNA and LMO4 protein of B group were statistically significantly lower than those of A group(P<0.05).The number of cell invasion of C group was statistically significantly more than that of A group，that of B group was statistically significantly less than that of A group，that of D group was statistically significantly less than that of C group，but was statistically significantly more than that of B group(P<0.05).After 24 hours of culture，the cell migration distance of C group was statistically significantly longer than that of A group，that of B group was statistically significantly shorter than that of A group，that of D group was statistically significantly shorter than that of C group，but was statistically significantly longer than that of B group(P<0.05).The relative expression quantity of N-cadherin protein and Vimentin protein of C group were statistically significantly higher than those of A group，while that of E-cadherin protein of C group was statistically significantly lower than that of A group(P<0.05)；the relative expression quantity of N-cadherin protein and Vimentin protein of B group were statistically significantly lower than those of A group，while that of E-cadherin protein of B group was statistically significantly higher than that of A group(P<0.05)；the relative expression quantity of N-cadherin protein and Vimentin protein of D group were statistically significantly lower than those of C group，while that of E-cadherin protein of D group was statistically significantly higher than that of A group(P<0.05)；the relative expression quantity of N-cadherin protein and Vimentin protein were statistically significantly higher than those of B group，while that of E-cadherin protein of D group was statistically significantly lower than that of B group(P<0.05).ConclusionLMO4 gene silencing can reverse the Snail-induced EMT of non-small cell lung cancer A549 cells.

【Key words】Carcinoma，non-small-cell lung；LMO4；Snail；Epithelial-mesenchymal transition

基金項目：國家自然科學基金(81301939)；博士后基金(2015M570697)

通信作者：王雯珺，510120廣東省廣州市，呼吸疾病國家重點實驗室，廣州醫(yī)科大學第一附屬醫(yī)院，廣州醫(yī)科大學；E-mail：wwj01@foxmail.com

【中圖分類號】R 730.26

【文獻標識碼】A

doi:10.3969/j.issn.1008-5971.2016.05.013

(收稿日期：2015-12-16；修回日期：2016-04-28)