李金輝 陳 翔 顏秀娟 李 海 陳勝良

上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院南院消化內(nèi)科(201112)

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·論 著·

MicroRNA-129通過(guò)靶向調(diào)控HMGA2抑制胃癌細(xì)胞增殖、侵襲

李金輝 陳 翔 顏秀娟 李 海 陳勝良*

上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院南院消化內(nèi)科(201112)

背景：越來(lái)越多的研究表明microRNA在胃癌發(fā)生、發(fā)展中起重要作用。有研究表明miR-129在胃癌組織中表達(dá)異常，但其對(duì)胃癌細(xì)胞增殖、侵襲的作用仍不明確。目的：探討miR-129在胃癌組織和胃癌細(xì)胞株中的表達(dá)及其影響胃癌細(xì)胞增殖和侵襲的機(jī)制。方法：收集82例胃癌組織及其相應(yīng)癌旁組織，培養(yǎng)人胃黏膜上皮細(xì)胞株和不同的胃癌細(xì)胞株，以qPCR法檢測(cè)miR-129表達(dá)。將miR-129 mimic或miR-NC轉(zhuǎn)染胃癌SGC-7901細(xì)胞后，轉(zhuǎn)染HMGA2過(guò)表達(dá)質(zhì)粒。以克隆形成實(shí)驗(yàn)觀(guān)察細(xì)胞增殖情況，Transwell小室法檢測(cè)細(xì)胞侵襲情況，Pearson相關(guān)分析評(píng)估m(xù)iR-129表達(dá)與HMGA2表達(dá)的相關(guān)性，熒光素酶實(shí)驗(yàn)檢測(cè)熒光素酶活性，qPCR和蛋白質(zhì)印跡法分別檢測(cè)miR-129、HMGA2 mRNA和蛋白表達(dá)。結(jié)果：與癌旁組織相比，胃癌組織中miR-129表達(dá)明顯下降(P<0.05)；與人胃黏膜上皮細(xì)胞株GES-1相比，各胃癌細(xì)胞株中miR-129表達(dá)明顯下降(P<0.05)。與miR-NC組相比，miR-129 mimic組SGC-7901細(xì)胞增殖、侵襲能力均明顯下降(P<0.05)。胃癌組織中HMGA2 mRNA表達(dá)明顯增加(P<0.05)，并與miR-129表達(dá)呈負(fù)相關(guān)(r=-0.543 9，P<0.01)。野生型miR-129 mimic組熒光素酶活性顯著低于miR-NC組(P<0.05)；轉(zhuǎn)染miR-129 mimic后HMGA2 mRNA和蛋白表達(dá)顯著降低(P<0.05)。與miR-129+陰性對(duì)照組相比，miR-129 mimic+HMGA2組細(xì)胞增殖、侵襲能力明顯升高(P<0.05)。結(jié)論：miR-129在胃癌組織和細(xì)胞中低表達(dá)，其可通過(guò)下調(diào)HMGA2抑制SGC-7901細(xì)胞的增殖和侵襲。

胃腫瘤； 微RNAs； HMGA2蛋白； 細(xì)胞增殖； 細(xì)胞侵襲

胃癌是最常見(jiàn)的惡性腫瘤之一，盡管其治療已取得一定的進(jìn)展，但5年死亡率仍很高，尤其是進(jìn)展期胃癌[1]。因此，深入了解胃癌發(fā)生、發(fā)展過(guò)程中的分子調(diào)控機(jī)制，有望為其治療提供新策略。MicroRNA(miRNA)是一類(lèi)長(zhǎng)度為21～25個(gè)核苷酸的單鏈RNA，屬于非編碼蛋白R(shí)NA，廣泛存在于生物界[2]。研究發(fā)現(xiàn)，miRNA在細(xì)胞增殖、凋亡、分化等過(guò)程中起重要作用[3]，從而參與腫瘤的發(fā)生、發(fā)展。大量研究表明miRNA參與胃癌的進(jìn)展、侵襲和轉(zhuǎn)移[4]。本課題的前期研究[5]發(fā)現(xiàn)miR-143在胃癌組織中表達(dá)下調(diào)，而上調(diào)miR-143能抑制胃癌細(xì)胞的增殖、侵襲能力。miR-129在多種腫瘤中異常表達(dá)并具有類(lèi)似抑癌基因的作用[6]。但有關(guān)其在胃癌中表達(dá)和作用的研究尚不多見(jiàn)。本研究通過(guò)檢測(cè)胃癌組織和胃癌細(xì)胞株中miR-129的表達(dá)，并進(jìn)行靶基因預(yù)測(cè)，旨在探索 miR-129對(duì)胃癌細(xì)胞增殖、侵襲的影響及其可能的機(jī)制，以期為胃癌治療提供新的靶點(diǎn)。

資料與方法

一、資料來(lái)源

收集 2013 年9月—2015年12月上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院南院82例胃癌組織和對(duì)應(yīng)的癌旁組織，其中男48例，女34例，年齡37～79歲，平均(47.6±9.3)歲。本研究方案由上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院倫理委員會(huì)審核通過(guò)，所有患者均簽署知情同意書(shū)。

二、細(xì)胞株、主要試劑

人胃黏膜上皮細(xì)胞株GES-1、胃癌細(xì)胞株SNU-16、BGC-823購(gòu)于上海蓋寧生物科技有限公司；胃癌細(xì)胞株MNK-45和SGC-7901保存于本實(shí)驗(yàn)室。RNA提取試劑、Lipofectamine?2000(Invitrogen公司)；逆轉(zhuǎn)錄試劑盒、SYBR Green熒光染料試劑盒[寶生物工程(大連)有限公司]；兔抗人HMGA2、GAPDH抗體(英國(guó)Abcam公司)；miR-129 mimic、陰性對(duì)照miR-NC和HMGA2過(guò)表達(dá)質(zhì)粒均由上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司合成；辣根過(guò)氧化物酶標(biāo)記的山羊抗兔IgG(H+L)(碧云天生物技術(shù)研究所)；pGL3-Promoter質(zhì)粒和pRL-TK質(zhì)粒以及雙熒光素酶報(bào)告基因檢測(cè)試劑盒(Promega公司)。

三、研究方法

1. 細(xì)胞培養(yǎng)和轉(zhuǎn)染：各細(xì)胞均用含10%胎牛血清的DMEM培養(yǎng)液，置于37 ℃、5% CO2的培養(yǎng)箱中培養(yǎng)。當(dāng)細(xì)胞生長(zhǎng)至融合度為80%～90%時(shí)，胰蛋白酶消化，常規(guī)傳代培養(yǎng)。選取對(duì)數(shù)生長(zhǎng)期細(xì)胞，按試劑盒說(shuō)明書(shū)進(jìn)行轉(zhuǎn)染。將5×105個(gè)/孔SGC-7901細(xì)胞接種于6孔板，生長(zhǎng)至60%～70%密度時(shí)，以100 μmol/L含有miR-129 mimic質(zhì)?；騧iR-NC、HMGA2過(guò)表達(dá)質(zhì)?；蚱潢幮詫?duì)照(vector)質(zhì)粒的脂質(zhì)體轉(zhuǎn)染6 h后換液，在普通培養(yǎng)基中繼續(xù)培養(yǎng) 24 h，用qPCR檢測(cè)轉(zhuǎn)染效率，再行后續(xù)實(shí)驗(yàn)。

2. qPCR法：提取組織或細(xì)胞中的總RNA，逆轉(zhuǎn)錄成cDNA。分別以U6和GAPDH作為內(nèi)參，各基因引物序列見(jiàn)表1。PCR反應(yīng)體系為25 μL，反應(yīng)條件：94 ℃ 15 min； 94 ℃ 20 s，60 ℃ 30 s，72 ℃ 30 s，共循環(huán)35次；72 ℃ 8 min。獨(dú)立重復(fù)實(shí)驗(yàn)3次，繪制擴(kuò)增曲線(xiàn)，目的基因的表達(dá)采用2-ΔΔCt法計(jì)算。

表1 各基因PCR引物序列

3. 蛋白質(zhì)印跡法檢測(cè)HMGA2表達(dá)：提取各組細(xì)胞總蛋白。BCA法定量蛋白，行電泳后轉(zhuǎn)移至PVDF膜。10%山羊血清封閉1 h，加入兔抗人HMGA2多克隆抗體(工作濃度1∶500)或GAPDH抗體(工作濃度1∶1 000)，4 ℃孵育過(guò)夜，TBST洗膜，加入辣根過(guò)氧化物酶標(biāo)記的山羊抗兔二抗孵育2 h。ECL法顯影，以Quantity One 4.4軟件分析條帶灰度。

4. 克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力：取轉(zhuǎn)染后的SGC-7901細(xì)胞，以1 000個(gè)/孔的密度接種于6孔板，培養(yǎng)箱中靜止培養(yǎng)2周，至肉眼可見(jiàn)細(xì)胞克隆時(shí)終止培養(yǎng)。棄培養(yǎng)液，PBS洗滌3次，4%多聚甲醛固定，0.1%結(jié)晶紫染色；計(jì)數(shù)細(xì)胞克隆數(shù)目。

5. Transwell小室法檢測(cè)細(xì)胞侵襲能力：轉(zhuǎn)染48 h 后取各組細(xì)胞，以5×104/孔接種于Matrigel-Transwell小室，小室內(nèi)采用無(wú)血清DMEM 300 μL培養(yǎng)基進(jìn)行培養(yǎng)，下室加入500 μL 10%胎牛血清培養(yǎng)液。培養(yǎng)24 h后用棉簽輕輕擦去上層細(xì)胞，取出小室。4%多聚甲醛固定，0.1%結(jié)晶紫染色，PBS清洗。熒光顯微鏡下隨機(jī)選取5個(gè)視野(×200)，觀(guān)察侵襲細(xì)胞并計(jì)數(shù)，實(shí)驗(yàn)重復(fù)3次。

6. 熒光素酶實(shí)驗(yàn)：將SGC-7901細(xì)胞以每孔5×104/孔接種于24孔板，次日轉(zhuǎn)染miR-129 mimic或miR-NC，第3天共轉(zhuǎn)染0.2 μg pGL3-HMGA2-3’-UTR-WT(野生型)或pGL3-HMGA2-3’-UTR-MUT(突變型)和pRL-TK質(zhì)粒，繼續(xù)培養(yǎng)48 h后進(jìn)行檢測(cè)，計(jì)算相對(duì)熒光素酶活性。相對(duì)熒光素酶活性=螢火蟲(chóng)熒光素酶活性值/海腎熒光素酶活性值。

四、統(tǒng)計(jì)學(xué)分析

結(jié) 果

一、miR-129在胃癌組織和細(xì)胞株中的表達(dá)

與癌旁組織相比，胃癌組織中miR-129表達(dá)明顯下降(P<0.05)(圖1A)。與人胃黏膜上皮細(xì)胞株GES-1相比，各胃癌細(xì)胞株中miR-129表達(dá)均明顯下降(P<0.05)(圖1B)。

二、miR-129表達(dá)對(duì)SGC-7901細(xì)胞增殖和侵襲的影響

qPCR法顯示，與空白對(duì)照組和miR-NC組相比，miR-129 mimic組SGC-7901細(xì)胞中miR-129表達(dá)明顯增加(P<0.01)(圖2A)?？寺⌒纬蓪?shí)驗(yàn)表明，miR-129表達(dá)上調(diào)后SGC-7901細(xì)胞增殖能力較空白對(duì)照組和miR-NC組明顯下降(P<0.05)(圖2B)。Transwell實(shí)驗(yàn)發(fā)現(xiàn)，miR-129表達(dá)上調(diào)后，細(xì)胞侵襲能力明顯下降(P<0.01)(圖2C)。

三、miR-129靶基因的預(yù)測(cè)和驗(yàn)證

與癌旁組織相比，胃癌組織中HMGA2 mRNA表達(dá)明顯升高(P<0.05)(圖3A)。Pearson相關(guān)分析表明胃癌組織中miR-129表達(dá)與HMGA2 mRNA表達(dá)呈負(fù)相關(guān)(r=-0.543 9，P<0.001)(圖3B)。利用miRBase、miTarget和TargetScanS等軟件行生物學(xué)分析，結(jié)果表明HMGA2 mRNA含有可與miR-129結(jié)合的3’-UTR(圖3C)。構(gòu)建并轉(zhuǎn)染含有HMGA2 3’-UTR的報(bào)告質(zhì)粒后發(fā)現(xiàn)，野生型miR-129 mimic組的熒光素酶活性顯著低于miR-NC組(P<0.05)，而突變型中兩組無(wú)明顯差異(P>0.05)(圖3D)。qPCR和蛋白質(zhì)印跡法表明，與空白對(duì)照組和miR-NC組相比，miR-129 mimic組HMGA2 mRNA和蛋白表達(dá)均明顯下降(P<0.05)(圖3E、3F)。說(shuō)明HMGA2在胃癌細(xì)胞中可能是miR-129的靶基因。

四、HMGA2對(duì)miR-129介導(dǎo)的SGC-7901細(xì)胞抑制作用的影響

與miR-129 mimic+vector組相比，miR-129 mimic+HMGA2組SGC-7901細(xì)胞HMGA2 mRNA和蛋白表達(dá)均明顯增加(P<0.05)(圖4A、4B)，細(xì)胞增殖和侵襲能力均明顯增強(qiáng)(P<0.05)(圖4C、4D)。

A：胃癌組織和癌旁組織中miR-129表達(dá)；B：不同細(xì)胞株中miR-129表達(dá)

A：miR-129表達(dá)(qPCR法)；B：細(xì)胞增殖能力(克隆形成實(shí)驗(yàn))；C：細(xì)胞侵襲能力(Transwell實(shí)驗(yàn))

A：胃癌組織中HMGA2 mRNA表達(dá)(qPCR法)；B：胃癌組織中miR-129表達(dá)與HMGA2表達(dá)的相關(guān)性分析；C：生物信息學(xué)軟件預(yù)測(cè)miR-129與HMGA2基因的結(jié)合位點(diǎn)；D：熒光素酶活性；E：HMGA2 mRNA表達(dá)(qPCR法)；F：HMGA2蛋白表達(dá)(蛋白質(zhì)印跡法)

圖3 HMGA2是miR-129的作用靶點(diǎn)

討 論

腫瘤的局部侵襲和遠(yuǎn)處轉(zhuǎn)移限制了手術(shù)運(yùn)用，因此降低腫瘤細(xì)胞的侵襲、轉(zhuǎn)移能力有重要的臨床意義。近年大量研究表明miRNA在腫瘤的發(fā)生發(fā)展、早期診斷、腫瘤細(xì)胞侵襲和轉(zhuǎn)移等方面均發(fā)揮重要作用[7]。目前已發(fā)現(xiàn)miR-129在多種腫瘤組織或細(xì)胞株中的表達(dá)明顯下降，包括腎癌、結(jié)直腸癌、肝癌、乳腺癌等[8-10]。miR-129作為抑癌因子，能抑制細(xì)胞增殖，促進(jìn)細(xì)胞凋亡，從而抑制腫瘤的進(jìn)展[11]。近年多項(xiàng)研究[12-13]發(fā)現(xiàn)miR-129啟動(dòng)子區(qū)和5’-UTR包含CpG島，其過(guò)度甲基化可引起miR-129表達(dá)下降，從而誘導(dǎo)胃癌的發(fā)生。且這一甲基化過(guò)程可能與胃癌患者化療耐藥有關(guān)[14]。順鉑耐藥的胃癌組織和細(xì)胞中miR-129表達(dá)下降，而過(guò)表達(dá)miR-129可使胃癌細(xì)胞重新恢復(fù)對(duì)順鉑的敏感性[15]。然而，有關(guān)miR-129對(duì)胃癌細(xì)胞增殖、侵襲能力的研究甚少。最近一項(xiàng)研究[16]發(fā)現(xiàn)miR-129在胃癌患者組織和外周血中表達(dá)均下降，促進(jìn)了胃癌細(xì)胞的增殖、遷移能力，該作用可能通過(guò)下游靶基因白細(xì)胞介素8(IL-8)而實(shí)現(xiàn)的。本研究發(fā)現(xiàn)miR-129表達(dá)在胃癌組織中下調(diào)，在胃癌細(xì)胞株中亦明顯下降；而過(guò)表達(dá)miR-129后，SGC-7901細(xì)胞的增殖、侵襲能力受到抑制，這與先前的報(bào)道相符。然而，miRNA對(duì)基因的調(diào)節(jié)作用是一個(gè)復(fù)雜的網(wǎng)絡(luò)系統(tǒng)，單一miRNA分子往往參與了多個(gè)目標(biāo)基因的調(diào)控。本研究應(yīng)用生物學(xué)信息預(yù)測(cè)軟件對(duì)miR-129的靶基因進(jìn)行預(yù)測(cè)，最終定位到HMGA2基因。

A：HMGA2 mRNA表達(dá)(qPCR法)；B：HMGA2蛋白表達(dá)(蛋白質(zhì)印跡法)；C：細(xì)胞增殖能力(克隆形成實(shí)驗(yàn))；D：細(xì)胞侵襲能力(Transwell實(shí)驗(yàn))

HMGA2是一類(lèi)非組蛋白染色體蛋白，可通過(guò)改變?nèi)旧|(zhì)結(jié)構(gòu)來(lái)調(diào)節(jié)轉(zhuǎn)錄[17]。HMGA2在胚胎發(fā)育時(shí)大量表達(dá)，此后逐漸下降并維持較低水平表達(dá)，提示其在胚胎發(fā)育的細(xì)胞增殖分化過(guò)程中發(fā)揮重要作用。目前研究發(fā)現(xiàn)HMGA2在各種惡性腫瘤如胃癌中過(guò)表達(dá)并與腫瘤預(yù)后息息相關(guān)[18]。Jun等[19]亦發(fā)現(xiàn)，胃癌中HMGA2表達(dá)明顯上調(diào)并與患者的總生存率呈負(fù)相關(guān)。這一作用可能與HMGA2能促進(jìn)胃癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化(EMT)有關(guān)[20]。過(guò)表達(dá)HMGA2能促進(jìn)胃癌細(xì)胞增殖和侵襲能力[21]，而下調(diào)HMGA2表達(dá)能通過(guò)減輕EMT來(lái)抑制胃癌細(xì)胞的增殖、侵襲能力[22]。本研究發(fā)現(xiàn)，HMGA2表達(dá)在胃癌組織中明顯增加，且與miR-129表達(dá)呈負(fù)相關(guān)。進(jìn)一步行細(xì)胞實(shí)驗(yàn)發(fā)現(xiàn)，miR-129 mimic干預(yù)SGC-7901細(xì)胞后HMGA2表達(dá)下降，熒光素酶實(shí)驗(yàn)結(jié)果表明miR-129可與HMGA2 3’-UTR結(jié)合，抑制HMGA2表達(dá)?；匮a(bǔ)實(shí)驗(yàn)結(jié)果顯示HMGA2參與了miR-129對(duì)胃癌細(xì)胞的抑制作用。推測(cè)miR-129能通過(guò)下調(diào)HMGA2表達(dá)來(lái)抑制胃癌細(xì)胞的增殖、侵襲能力。此外，研究[20]發(fā)現(xiàn)HMGA2敲除后，EMT標(biāo)記物發(fā)生逆轉(zhuǎn)，認(rèn)為其促進(jìn)胃癌侵襲轉(zhuǎn)移的作用可能是通過(guò)促進(jìn)EMT而實(shí)現(xiàn)的。最近一項(xiàng)細(xì)胞和動(dòng)物實(shí)驗(yàn)[23]發(fā)現(xiàn)HMGA2促進(jìn)EMT的作用主要是通過(guò)調(diào)節(jié)下游靶基因Twist相關(guān)蛋白1(TWIST1)完成的。由此可見(jiàn)，HMGA2有望成為胃癌治療的潛在靶點(diǎn)。

綜上所述，胃癌中miR-129表達(dá)下調(diào)，miR-129可靶向調(diào)控HMGA2表達(dá)，從而抑制胃癌細(xì)胞的增殖、侵襲能力，進(jìn)一步證實(shí)了miR-129在胃癌發(fā)生、發(fā)展中的重要作用，為胃癌的治療提供了新思路。

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(2017-04-06收稿；2017-04-18修回)

MicroRNA-129 Inhibits Gastric Cancer Cells Proliferation and Invasion by Targeting HMGA2

LI Jinhui, CHEN Xiang, YAN Xiujuan, LI Hai, CHEN Shengliang.

Division of Gastroenterology and Hepatology, South Campus, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (201112)

CHEN Shengliang, Email: chenslmdrj@sina.com

Stomach Neoplasms; MicroRNAs; HMGA2 Protein; Cell Proliferation; Cell Invasion

10.3969/j.issn.1008-7125.2017.07.002

*本文通信作者，Email: chenslmdrj@sina.com

Background: More and more evidences suggest that microRNA plays an important role in the development of gastric cancer (GC). Expression of miR-129 in GC tissues is found abnormal, however, the mechanism of miR-129 in cell proliferation and invasion is still undefined. Aims: To investigate the expression of miR-129 in GC tissue and GC cell lines and the mechanism of miR-129 in proliferation and invasion of SGC-7901 cells. Methods: Eighty-two GC tissues and corresponding paracancerous tissues were collected, human gastric epithelial cell line and different GC cell lines were cultured, qPCR was conducted to assess miR-129 expression. SGC-7901 cells were transfected with miR-129 mimic or miR-NC, and then were transfected with overexpressed HMGA2 plasmid. Colony formation assay was used to detect cell proliferation ability, and Transwell chamber was used to assess cell invasion ability. Pearson correlation analysis was used to analyze the correlation between miR-129 and HMGA2 mRNA expression. Luciferase assay was performed to determine the activity of luciferase. mRNA and protein expressions of miR-129, HMGA2 were determined by qPCR and Western blotting, respectively. Results: Compared with paracancerous tissues, expression of miR-129 was significantly decreased in GC tissues (P<0.05); when compared with human gastric epithelial cells, expression of miR-129 was significantly decreased in GC cell lines (P<0.05). Compared with miR-NC group, proliferation and invasion abilities of SGC-7901 cells were inhibited in miR-129 mimic group (P<0.05). HMGA2 mRNA expression in GC tissues was significantly upregulated (P<0.05), and was negatively correlated with miR-129 expression (r=-0.543 9,P<0.01). Luciferase activity in wild-type miR-129 mimic group was significantly lower than that in miR-NC group (P<0.05); mRNA and protein expressions of HMGA2 were decreased after transfection with miR-129 mimic (P<0.05). Compared with miR-129+vector group, proliferation and invasion of SGC-7901 cells were significantly increased in miR-129 mimic+HMGA2 group (P<0.05). Conclusions: The expression of miR-129 is decreased in GC tissue and cells; miR-129 inhibits SGC-7901 cells proliferation and invasion by negatively regulating HMGA2.