微小RNA在叉頭轉(zhuǎn)錄因子M1激活非小細(xì)胞肺癌上皮向間質(zhì)轉(zhuǎn)化中的作用

吳 艷， 鄒黎菲， 惠復(fù)新， 汪家坤

南京醫(yī)科大學(xué)附屬無(wú)錫人民醫(yī)院呼吸科，無(wú)錫 214023

論 著

微小RNA在叉頭轉(zhuǎn)錄因子M1激活非小細(xì)胞肺癌上皮向間質(zhì)轉(zhuǎn)化中的作用

吳 艷， 鄒黎菲， 惠復(fù)新， 汪家坤*

南京醫(yī)科大學(xué)附屬無(wú)錫人民醫(yī)院呼吸科，無(wú)錫 214023

目的：探討微小RNA(microRNA)在叉頭轉(zhuǎn)錄因子M1(Fox M1)激活非小細(xì)胞肺癌(non-small cell lung cancer, NSCLC)細(xì)胞上皮向間質(zhì)轉(zhuǎn)化(epithelial-mesenehymal transition，EMT)中的作用。方法：將Fox M1過(guò)表達(dá)質(zhì)粒，F(xiàn)ox M1-shRNA，miR-539、miR-485-5p模擬物及其抑制物轉(zhuǎn)染至人NSCLC細(xì)胞株中，應(yīng)用Western印跡和real-time PCR檢測(cè)細(xì)胞株中Fox M1蛋白和mRNA及miR-539、miR-485-5p的表達(dá)。同時(shí)應(yīng)用CCK-8和細(xì)胞遷移實(shí)驗(yàn)觀察Fox M1、miR-539和 miR-485-5p對(duì)NSCLC細(xì)胞的增殖和侵襲功能的影響。應(yīng)用熒光素酶報(bào)告基因?qū)嶒?yàn)檢測(cè)miR-539和 miR-485-5p與EMT調(diào)控蛋白ZEB1和Snail1的關(guān)系。結(jié)果：Fox M1過(guò)表達(dá)下調(diào)NSCLC 細(xì)胞中miR-539、miR-485-5p，轉(zhuǎn)染Fox M1-shRNA后NSCLC細(xì)胞中 miR-539、miR-485-5p升高。MiR-539和miR-485-5p抑制Fox M1對(duì)NSCLC細(xì)胞增殖和侵襲的促進(jìn)作用。NSCLC細(xì)胞中miR-539對(duì)EMT調(diào)控蛋白ZEB1，miR-485-5p對(duì)EMT調(diào)控蛋白Snail1的表達(dá)有抑制作用。結(jié)論：miR-539和miR-485-5p能抑制NSCLC細(xì)胞中Fox M1-EMT通路，從而影響NSCLC細(xì)胞的增殖和侵襲，抑制NSCLC的遠(yuǎn)處轉(zhuǎn)移。

叉頭轉(zhuǎn)錄因子M1；上皮向間質(zhì)轉(zhuǎn)化；非小細(xì)胞肺癌；微小RNA

惡性腫瘤是我國(guó)城市居民死亡的首位原因，占全部死亡總數(shù)的25%，肺癌已替代肝癌成為我國(guó)惡性腫瘤的首位死亡原因。肺癌的遠(yuǎn)端轉(zhuǎn)移是肺癌患者的主要死亡原因[1]。肺癌中非小細(xì)胞肺癌(non-small cell lung cancer, NSCLC)約占所有肺癌的85%，其臨床癥狀重，預(yù)后差[2-3]。目前，應(yīng)用于臨床的靶向治療藥物可使NSCLC患者的生存期明顯延長(zhǎng)，生活質(zhì)量顯著提高。但這些藥物治療的靶點(diǎn)局限，且僅對(duì)小部分患者有效，因此急需尋找新的治療靶點(diǎn)。Fox (forehead box) M1是Fox轉(zhuǎn)錄因子家族的成員。研究[4]表明，F(xiàn)ox M1與NSCLC患者的不良預(yù)后密切相關(guān)。Fox M1的過(guò)表達(dá)能促進(jìn)NSCLC腫瘤細(xì)胞增殖、侵襲和遷移[5]。而EMT在多種腫瘤的轉(zhuǎn)移中發(fā)揮重要作用[6]。研究[7]發(fā)現(xiàn)，上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition，EMT)在Fox M1促進(jìn) NSCLC腫瘤細(xì)胞遷移和侵襲能力中亦發(fā)揮關(guān)鍵作用。研究[8-9]證實(shí)，miRNA在NSCLC中具有重要的調(diào)節(jié)作用。miR-663a在NSCLC組織中下調(diào)，并且通過(guò)靶向JunD作為腫瘤抑制劑起作用[10]。MiR-223通過(guò)靶向胰島素樣生長(zhǎng)因子-1受體增強(qiáng)NSCLC細(xì)胞對(duì)厄洛替尼的敏感性[11]。Fox M1-EMT是涉及多個(gè)信號(hào)通路和分子調(diào)節(jié)機(jī)制的復(fù)雜過(guò)程，這個(gè)調(diào)控網(wǎng)絡(luò)中不僅包含一些蛋白分子、調(diào)控因子信號(hào)的調(diào)控，還受到miRNA的調(diào)控。因此，本研究通過(guò)觀察miR-539、miR-485-5p對(duì)NSCLC細(xì)胞中Fox M1-EMT信號(hào)通路的影響，為NSCLC靶向治療提供新的思路。

1 材料與方法

1.1 細(xì)胞培養(yǎng) 人NSCLC細(xì)胞系A(chǔ)549和H1299購(gòu)自中國(guó)科學(xué)院上海細(xì)胞庫(kù)(中國(guó)上海)，并用含10%胎牛血清(FBS，Hyclone，美國(guó))的Dulbecco改良Eagle培養(yǎng)基(DMEM，Invitrogen，美國(guó))在37℃、5%CO2的濕潤(rùn)培養(yǎng)箱中培養(yǎng)。

1.2 細(xì)胞轉(zhuǎn)染 Fox M1過(guò)表達(dá)質(zhì)粒、Fox M1抑制劑(Fox M1-shRNA，Genepharma，中國(guó))，使用Lipofectamine 2000(Invitrogen，美國(guó))根據(jù)說(shuō)明書(shū)分別轉(zhuǎn)染至NSCLC細(xì)胞中。NSCLC細(xì)胞分別轉(zhuǎn)染miR-539、miR-485-5p 模擬物(mimic)和對(duì)照miRNA(Genepharma，中國(guó))，應(yīng)用Lipofectamine 2000以50 nmol/L濃度轉(zhuǎn)染。同樣，miR-539、miR-485-5p抑制劑(inhibitor)和對(duì)照(Genepharma，中國(guó))根據(jù)說(shuō)明書(shū)使用Lipofectamine 2000以100 nmol/L濃度轉(zhuǎn)染入NSCLC細(xì)胞中。

1.3 qRT-PCR 用TRIzol試劑(Invitrogen，美國(guó))從細(xì)胞中提取總RNA，并通過(guò)NanoDrop ND-1000分光光度計(jì)(Agilent，美國(guó))測(cè)量濃度。ABI 7500系統(tǒng)(Applied Biosystems，美國(guó))進(jìn)行qRT-PCR。反轉(zhuǎn)錄和qRT-PCR擴(kuò)增用具有g(shù)DNA Eraser(TaKaRa，日本)和SYBR Prime Script RT-PCR試劑盒(TaKaRa，日本)的PrimeScript RT試劑盒。MiRNA的反轉(zhuǎn)錄和qRT-PCR擴(kuò)增使用miDETECA TrackTMmiRNA qRT-PCR入門(mén)試劑盒(Rib Bio，中國(guó))。以β-actin作為內(nèi)參，miRNA以U6作為內(nèi)參。每個(gè)實(shí)驗(yàn)進(jìn)行至少3次。qRT-PCR的引物如表1。

表1 qRT-PCR的引物序列

1.4 免疫印跡法 將NSCLC細(xì)胞裂解并用1×SDS-PAGE上樣緩沖液提取到蛋白質(zhì)中。使用BCA蛋白測(cè)定試劑盒(Beyotime Institute of Biotechnology，中國(guó))測(cè)定蛋白質(zhì)濃度。在10%SDS-PAGE凝膠上分離等量的蛋白質(zhì)，然后轉(zhuǎn)移至PVDF膜。在用5%脫脂乳封閉2 h后，用如下的第一抗體溫育膜：Fox M1(1∶250，Santa Cruz，美國(guó))和β-actin(1∶1 000，Santa Cruz，美國(guó))。

1.5 CCK-8測(cè)定 使用細(xì)胞計(jì)數(shù)試劑盒-8(CCK-8)測(cè)定試劑盒(Dojindo，日本)測(cè)定細(xì)胞增殖。將轉(zhuǎn)染的細(xì)胞以2×103個(gè)細(xì)胞/孔的密度接種在96孔板中；第2 d向每個(gè)孔中加入10 μL CCK-8溶液。將細(xì)胞孵育70 min，并使用酶聯(lián)免疫吸附測(cè)定讀數(shù)器(Dasit，意大利)測(cè)試450 nm處的光密度值，共6 d，每個(gè)實(shí)驗(yàn)進(jìn)行至少3次，并分析結(jié)果的平均值。

1.6 細(xì)胞遷移測(cè)定 將無(wú)血清培養(yǎng)基中的轉(zhuǎn)染的NSCLC細(xì)胞以合適的密度接種在具有8 mm直徑(Corning，美國(guó))的24孔格式的Transwell室的上部。在底室中，加入800 μL含有10%FBS的正常DMEM培養(yǎng)基，并將培養(yǎng)箱在37℃、5%CO2下培養(yǎng)24～48 h。用棉簽輕輕擦去上部的細(xì)胞，并將遷移的細(xì)胞用0.05%結(jié)晶紫染色30 min。在顯微鏡下5個(gè)隨機(jī)視野中計(jì)數(shù)遷移的細(xì)胞，每個(gè)實(shí)驗(yàn)進(jìn)行至少3次，分析結(jié)果的平均值。

1.7 熒光素酶報(bào)告基因測(cè)定 將含有完整miR-539識(shí)別序列的ZEB1的3′-UTR和含有完整miR-485-5p識(shí)別序列的Snail13′-UTR克隆到pmiR-RB-ReportTM(Rib Bio，中國(guó))中。將細(xì)胞(5×104)接種在24孔板中并孵育24 h后進(jìn)行轉(zhuǎn)染。應(yīng)用Lipofectamine 2000轉(zhuǎn)染包含miRNA(miR-539和miR-485-5p)模擬物以及陰性對(duì)照的3′-UTR(或3′-UTR-突變體)的螢火蟲(chóng)熒光素酶構(gòu)建體。轉(zhuǎn)染48 h后裂解細(xì)胞，提取蛋白，用Dual-Luciferase Reporter System(Promega，中國(guó))測(cè)量熒光信號(hào)變化。

1.8 統(tǒng)計(jì)學(xué)處理 采用GraphPad Prism 6.0進(jìn)行統(tǒng)計(jì)分析。兩組間的差異通過(guò)Student’sttest進(jìn)行檢驗(yàn)。檢驗(yàn)水準(zhǔn)(α)為0.05。

2 結(jié) 果

2.1 Fox M1下調(diào)NSCLC 細(xì)胞中miR-539、miR-485-5p mRNA的水平 通過(guò)轉(zhuǎn)染過(guò)表達(dá)Fox M1的質(zhì)粒及Fox M1的shRNA，結(jié)果顯示不同NSCLCs細(xì)胞株Fox M1的mRNA及蛋白水平均能穩(wěn)定地顯示Fox M1高表達(dá)及低表達(dá)特性(圖1A～1D)。MiR-539、miR-485-5p mRNA水平在不同的NSCLC細(xì)胞系中均表現(xiàn)為在Fox M1高表達(dá)時(shí)降低，在Fox M1被抑制時(shí)升高(圖1E、1F)。

圖1 NSCLC細(xì)胞系A(chǔ)549和H1299中Fox M1過(guò)表達(dá)或表達(dá)抑制對(duì)miR-539、miR-485-5p mRNA表達(dá)的影響

2.2 miR-539和miR-485-5p抑制Fox M1對(duì)NSCLC細(xì)胞增殖和侵襲的影響 本研究通過(guò)轉(zhuǎn)染miR-539和miR-485-5p，使NSCLC細(xì)胞過(guò)表達(dá)miR-539和miR-485-5p，并檢測(cè)CCK-8及細(xì)胞遷移能力來(lái)觀察miR-539和miR-485-5p的過(guò)表達(dá)對(duì)NSCLC細(xì)胞增殖和侵襲的影響，發(fā)現(xiàn)miR-539和miR-485-5p過(guò)表達(dá)抑制NSCLC細(xì)胞增殖和侵襲(圖2A、2B)。相反，抑制miR-539和miR-485-5p增強(qiáng)了NSCLC細(xì)胞增殖和侵襲的促進(jìn)作用(圖2C、2D)。在過(guò)表達(dá)Fox M1的細(xì)胞中，miR-539和miR-485-5p過(guò)表達(dá)抑制Fox M1對(duì)NSCLC細(xì)胞增殖和侵襲的促進(jìn)作用，抑制miR-539和miR-485-5p的表達(dá)增強(qiáng)Fox M1對(duì)NSCLC細(xì)胞增殖和侵襲的促進(jìn)作用(圖3)。

圖2 miR-539和miR-485-5p對(duì)NSCLC細(xì)胞(H1299)增殖和侵襲能力的影響

A, B: miR-539和miR-485-5p對(duì)H1299細(xì)胞增殖的抑制作用；C, D: miR-539和miR-485-5p對(duì)H1299細(xì)胞遷移能力的抑制作用.NC: normal control; miR-539: miR-539 mimic; miR-485-5p: miR-485-5p mimic.*P<0.05, **P<0.01;n=3

2.3 NSCLC細(xì)胞中miR-539對(duì)ZEB1以及miR-485-5p對(duì) Snail1的調(diào)控 通過(guò)生物信息學(xué)方法，本研究發(fā)現(xiàn)miR-539 能結(jié)合 ZEB1的3′-UTR區(qū)，miR-485-5p能與Snail1的3′-UTR結(jié)合(圖4A)。為了了解miR-539和miR-485-5p是否能分別調(diào)控ZEB1和Snail1，我們通過(guò)基因轉(zhuǎn)染的途徑在NSCLC細(xì)胞中過(guò)表達(dá)或抑制miR-539和miR-485-5p時(shí)發(fā)現(xiàn)Fox M1的蛋白和mRNA水平升高或降低(圖4B～4E)。同時(shí)將3′-UTR ZEB1和Snail1含有野生型或突變型miR-539和miR-485-5p結(jié)合序列分別克隆到熒光素酶報(bào)告基因載體中并將載體同時(shí)轉(zhuǎn)染入細(xì)胞后，熒光素酶報(bào)告基因?qū)嶒?yàn)證明，miR-539和miR-485-5p能有效抑制野生型熒光素酶活性而對(duì)突變型熒光素酶活性無(wú)影響(圖4F)。

圖3 miR-539和miR-485-5p對(duì) Fox M1對(duì)NSCLC細(xì)胞(H1299)增殖和侵襲能力的抑制作用

A, B: miR-539和miR-485-5p抑制H1299細(xì)胞中Fox M1對(duì)H1299增殖的影響; C, D: miR-539和miR-485-5p抑制H1299細(xì)胞中Fox M1對(duì)H1299遷移能力的影響.a, d: Fox M1+NC; b: Fox M1+miR-539 mimic; c: Fox M1+miR-539 inhibitor; e: Fox M1+miR-485-5p mimic; f: Fox M1+miR-485-5p inhibitor.*P<0.05,**P<0.01;n=3

圖4 H1299細(xì)胞中miR-539對(duì)ZEB1以及miR-485-5p對(duì) Snail1的調(diào)控

A: miR-539 能結(jié)合 ZEB1的3′-UTR區(qū), miR-485-5p能與Snail13′-UTR 結(jié)合; B, C: miR-539 和miR-485-5p過(guò)表達(dá)對(duì)H1299細(xì)胞中Fox M1的蛋白水平(B)及mRNA水平的影響(C); D, E: miR-539 和 miR-485-5p的抑制對(duì)H1299細(xì)胞中Fox M1的蛋白水平(D)和mRNA水平(E)的影響; F: miR-539和miR-485-5p過(guò)表達(dá)能有效抑制野生型熒光素酶活性而對(duì)突變型熒光素酶活性無(wú)影響.*P<0.05,**P<0.01;n=3

3 討 論

miRNA 是一類(lèi)廣泛存在于動(dòng)植物和病毒中長(zhǎng)為20～25個(gè)核苷酸的內(nèi)源性小分子非編碼RNA。研究[12]發(fā)現(xiàn) miRNA與肺癌的類(lèi)型、進(jìn)展、患者預(yù)后及生存率密切相關(guān)。 MiRNA 可作為腫瘤抑制或促進(jìn)因子調(diào)控腫瘤細(xì)胞的增殖、凋亡、侵襲和癌性血管生成等。研究[13-16]發(fā)現(xiàn)miR-485-5p、miR-539在多種腫瘤中均有明顯下調(diào)，可作為腫瘤惡化的抑制因子。MiR-485-5p可能成為NSCLC早期診斷的標(biāo)志物[17]，而miR-539能增強(qiáng)順鉑對(duì)非小細(xì)胞肺癌耐藥株的殺傷作用[18]。在這項(xiàng)研究中，我們發(fā)現(xiàn)在NSCLC細(xì)胞中Fox M1上調(diào)EMT可能是通過(guò)抑制miR-539和miR-485-5p的表達(dá)發(fā)揮作用。

本研究發(fā)現(xiàn)，在NSCLC細(xì)胞株中Fox M1的表達(dá)抑制miR-539、 miR-485-5p。 miR-539、 miR-485-5p mRNA在Fox M1高表達(dá)時(shí)降低，在Fox M1表達(dá)被抑制時(shí)升高。功能實(shí)驗(yàn)發(fā)現(xiàn)miR-539和miR-485-5p過(guò)表達(dá)時(shí)NSCLC細(xì)胞的增殖和遷移能力減弱，miR-539和miR-485-5p被抑制時(shí)細(xì)胞的增殖和遷移能力增強(qiáng)。在過(guò)表達(dá)Fox M1的H1299細(xì)胞內(nèi)，miR-539和miR-485-5p能抑制由于Fox M1導(dǎo)致的細(xì)胞侵襲和增殖能力的增強(qiáng)。因此，miR-539和miR-485-5p可能是通過(guò)Fox M1來(lái)調(diào)控NSCLC細(xì)胞的功能。

研究[19]發(fā)現(xiàn)，F(xiàn)ox M1通過(guò)EMT促進(jìn) NSCLC腫瘤細(xì)胞遷移和侵襲。研究[20]表明，肺癌EMT過(guò)程受到多種miRNA調(diào)控，miR-138通過(guò)靶向GIT1和SEMA4C抑制非小細(xì)胞肺癌細(xì)胞的細(xì)胞增殖和逆轉(zhuǎn)EMT。MiR-145和miR-203通過(guò)抑制非小細(xì)胞肺癌細(xì)胞中的SMAD3抑制TGF-β誘導(dǎo)的EMT[21]，miR-338-3p通過(guò)靶向EMT抑制肺癌細(xì)胞的轉(zhuǎn)移[22]。然而miR-539和miR-485-5p是否能通過(guò)Fox M1-EMT途徑來(lái)調(diào)控NSCLC細(xì)胞功能尚未可知。多項(xiàng)研究[23-24]證實(shí)ZEB1和Snail1是調(diào)控EMT的重要蛋白。Fox M1能通過(guò)激活ZEB1和Snail1等促進(jìn)腫瘤細(xì)胞EMT過(guò)程[25-27]。實(shí)驗(yàn)通過(guò)生物信息分析發(fā)現(xiàn)，miR-539 能結(jié)合 ZEB1的3′-UTR區(qū)，miR-485-5p能與Snail13′-UTR 結(jié)合。熒光素酶報(bào)告基因?qū)嶒?yàn)發(fā)現(xiàn)，miR-539和miR-485-5p的過(guò)表達(dá)能分別抑制ZEB1和Snail1的表達(dá)，參與調(diào)控NSCLC細(xì)胞的Fox M1-EMT通路。

綜上所述，本研究證實(shí)miR-539和miR-485-5p能通過(guò)調(diào)控Fox M1-EMT通路來(lái)影響NSCLC腫瘤細(xì)胞增殖和侵襲能力，影響腫瘤的遠(yuǎn)處轉(zhuǎn)移，為臨床NSCLC的診治提供了新的理論依據(jù)。

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[本文編輯] 葉 婷， 曉 璐

The role of microRNAs in activation of epithelial-mesenchymal transition in non-small-cell lung cancer by forkhead box M1

WU Yan, ZOU Li-fei, HUI Fu-xin, WANG Jia-kun*

Department of Respiratory Medicine, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi 214023, Jiangsu, China

Objective：To investigate the role of microRNAs in Fox M1-induced non-small cell lung cancer (NSCLC) epithelial-mesenchymal transition (EMT).Methods：Over expression Fox M1plasmid, Fox M1-shRNA, miR-539, miR-485-5p mimics and inhibitor were transfected into NSCLC cells.The expression of Fox M1protein and mRNA, miR-539 and miR-485-5p were detected by Western blotting and real-time PCR.The CCK-8 and cell migration was used to observe the effect of Fox M1, miR-539 and miR-485-5p on the proliferation and invasion of NSCLC.Luciferase reporter assay was used to explore the relationship between miRNA( miR-539 and miR-485-5p) and EMT regulatory protein (ZEB1and Snail1).Results：Real-time RT-PCR and Western blotting showed Fox M1over expression inhibited the expression of miR-539, miR-485-5p, miR-539 and miR-485-5p in NSCLC cells were increased after transfected Fox M1-shRNA.MiR-539 and miR-485-5p suppressed enhancement of proliferation and invasion of NSCLC cells by Fox M1.miR-539 targeted and inhibited the translation of ZEB1,miR-485-5p targeted and inhibited the translation of Snail1in NSCLC cells.Conclusions：The mechanism of Fox M1up-regulation of EMT protein is to decrease the expression of miR-539 and miR-485-5p in NSCLC cells, thus to affect the proliferation and invasion of NSCLC cells, and to inhibit distant metastasis.

forkhead box M1; epithelial-mesenchymal transition; NSCLC; microRNA

2016-09-26[接受日期]2016-11-30

吳 艷，碩士，副主任醫(yī)師.E-mail: wuyanyangting@163.com

*通信作者(Corresponding author).Tel: 0510-85350137, E-mail: wwwztg90203@sina.com

10.12025/j.issn.1008-6358.2016.20160990

R 734.2

A