唐夏莉，　焦德敏，　陳　君，　王　劍，　陳清勇

(中國(guó)人民解放軍第一一七醫(yī)院呼吸內(nèi)科，浙江 杭州 310004)

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miRNA-126對(duì)肺癌A549細(xì)胞的增殖、遷移、侵襲及EGFR/AKT/mTOR信號(hào)通路的影響*

唐夏莉，焦德敏，陳君，王劍，陳清勇△

(中國(guó)人民解放軍第一一七醫(yī)院呼吸內(nèi)科，浙江 杭州 310004)

[摘要]目的： 探討微小RNA(microRNA,miRNA)-126對(duì)肺癌A549細(xì)胞功能的影響及相關(guān)的作用機(jī)制。方法: 利用脂質(zhì)體試劑Lipofectamine 2000將miRNA-126轉(zhuǎn)染入肺癌A549細(xì)胞中，采用real-time PCR法檢測(cè)轉(zhuǎn)染后各組細(xì)胞中miRNA-126的表達(dá)；MTT法檢測(cè)細(xì)胞活力；臺(tái)盼藍(lán)拒染實(shí)驗(yàn)檢測(cè)細(xì)胞存活數(shù)目；細(xì)胞集落培養(yǎng)實(shí)驗(yàn)觀察轉(zhuǎn)染后細(xì)胞集落形成；劃痕愈合實(shí)驗(yàn)檢測(cè)細(xì)胞的遷移能力；Transwell小室實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲能力；Western blot法檢測(cè)EGFR/AKT/mTOR通路中各蛋白水平的變化。結(jié)果: Real-time PCR結(jié)果顯示，轉(zhuǎn)染miRNA-126組細(xì)胞中的miRNA-126表達(dá)水平顯著高于陰性對(duì)照組和空白對(duì)照組(P<0.01)，而陰性對(duì)照組與空白對(duì)照組無(wú)顯著變化；A549細(xì)胞轉(zhuǎn)染miRNA-126模擬物后，細(xì)胞增殖和集落形成能力均呈明顯下降(P<0.01)；肺癌細(xì)胞的遷移和侵襲能力也受到明顯抑制(P<0.01)；Western blot結(jié)果顯示，轉(zhuǎn)染miRNA-126組細(xì)胞中EGFR/AKT/mTOR通路相關(guān)蛋白p-EGFR、p-AKT和p-mTOR的水平均有明顯降低(P<0.01)。結(jié)論: miRNA-126可以顯著降低肺癌A549細(xì)胞中p-EGFR、p-AKT和p-mTOR的蛋白水平，進(jìn)而可能抑制其細(xì)胞增殖和遷移侵襲能力。

[關(guān)鍵詞]微小RNA-126; EGFR/AKT/mTOR通路; 肺癌; 細(xì)胞增殖

肺癌是目前世界上最常見(jiàn)的惡性腫瘤，研究表明其發(fā)病率和死亡率逐年升高[1]。在肺癌的許多分類中，非小細(xì)胞肺癌(non-small cell lung cancer，NSCLC)約占肺癌總數(shù)的80%～85%[2]。肺癌早期診斷困難，大多確診已在晚期，因此迫切需要尋找一種靈敏度高、特異性強(qiáng)的早期肺癌診斷標(biāo)記。微小RNA(microRNA，miRNA)是近年來(lái)發(fā)現(xiàn)的一大類內(nèi)源性的非編碼單鏈小分子RNA。已有大量的研究表明microRNA參與腫瘤發(fā)生發(fā)展的全過(guò)程，在細(xì)胞增殖、凋亡、遷移等多個(gè)生物進(jìn)程中發(fā)揮著類似癌基因或抑癌基因的作用。如miRNA-34a促進(jìn)甲狀腺癌細(xì)胞增殖[3]，而miRNA-206則抑制肺癌細(xì)胞增殖和轉(zhuǎn)移[4]。本課題組在前期研究工作中利用miRNA表達(dá)譜芯片技術(shù)，發(fā)現(xiàn)miRNA-126在肺癌組織與癌旁組織中差異表達(dá)，并將miRNA-126轉(zhuǎn)染于肺癌A549細(xì)胞其增殖能力受到明顯抑制。因此，進(jìn)一步觀察miRNA-126在肺癌中的功能及其作用機(jī)制，不僅有助于肺癌的早期診斷，也有助于為肺癌的臨床治療提供有效的干預(yù)靶點(diǎn)，為發(fā)展新型抗肺癌基因藥物提供理論依據(jù)。

材料和方法

1材料

非小細(xì)胞肺癌A549細(xì)胞購(gòu)自中國(guó)科學(xué)院上海細(xì)胞所；has-miRNA-126-3p agomir引物序列、DMSO購(gòu)自Sigma；胰蛋白酶和RPMI-1640培養(yǎng)基購(gòu)自Gibco；四甲基偶氮唑鹽(methyl thiazolyl tetrazolium，MTT)、臺(tái)盼藍(lán)染色細(xì)胞存活率檢測(cè)試劑盒購(gòu)于碧云天生物公司；GAPDH、表皮生長(zhǎng)因子受體(epidermal growth factor receptor,EGFR)、p-EGFR、AKT、p-AKT、哺乳動(dòng)物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)和p-mTOR抗體購(gòu)自CST。倒置熒光顯微鏡購(gòu)自Leica; 凝膠成像系統(tǒng)和iMARK型全自動(dòng)酶標(biāo)儀購(gòu)自Bio-Rad。

2主要方法

2.1細(xì)胞的瞬時(shí)轉(zhuǎn)染轉(zhuǎn)染前1 d，接種適量的細(xì)胞于不含抗生素的培養(yǎng)基中，根據(jù)脂質(zhì)體Lipo-fectamine 2000的說(shuō)明書(shū)進(jìn)行細(xì)胞的轉(zhuǎn)染。分別用適量的Opti-MEM無(wú)血清培養(yǎng)液稀釋脂質(zhì)體和has-miR-126-3p agomir，室溫孵育5 min后混合，室溫靜置20 min，將混合液加入到每孔中，輕輕晃動(dòng)培養(yǎng)板混勻液體。將培養(yǎng)板放入培養(yǎng)箱中，6 h后替換為原培養(yǎng)基繼續(xù)培養(yǎng)。轉(zhuǎn)染帶熒光的FAM-NC時(shí)，所有操作均需避光操作，并以錫箔紙包被培養(yǎng)板放置培養(yǎng)箱中培養(yǎng)，轉(zhuǎn)染48 h后用PBS沖洗1次，熒光顯微鏡下觀察轉(zhuǎn)染效率。

2.2Real-time PCR檢測(cè)miRNA-126的表達(dá)各組A549細(xì)胞經(jīng)轉(zhuǎn)染48 h后，按照TRIzol說(shuō)明書(shū)中步驟抽提細(xì)胞總RNA，取RNA于紫外分光光度計(jì)下測(cè)A260/A280，比值處于1.8～2.1之間則符合純度要求，按miRNA逆轉(zhuǎn)錄試劑盒說(shuō)明配制逆轉(zhuǎn)錄試劑，逆轉(zhuǎn)錄合成cDNA，將合成的cDNA稀釋10倍，按real-time PCR說(shuō)明書(shū)配制反應(yīng)體系，每個(gè)樣本設(shè)3個(gè)復(fù)孔。PCR反應(yīng)步驟：95 ℃ 10 min；95 ℃ 15 s，60 ℃ 1 min，共40個(gè)循環(huán)。反應(yīng)以U6為內(nèi)參照。Has-miR-126-3p agomir的上游序列為5’-UCG UAC CGU GAG UAA UAA UGC G-3’，下游序列為5’-CAU UAU UAC UCA CGG UAC GAU U-3’。記錄各孔Ct值，取各孔平均值作為結(jié)果，并采用2-ΔΔCt法對(duì)結(jié)果進(jìn)行分析。

2.3MTT法檢測(cè)細(xì)胞活力取對(duì)數(shù)生長(zhǎng)期細(xì)胞以5.0×107/L的濃度接種于96孔細(xì)胞培養(yǎng)板中，每孔100 μL，置于37 ℃、5% CO2飽和濕度培養(yǎng)箱中培養(yǎng)24 h，待細(xì)胞貼壁后，按上述轉(zhuǎn)染方法轉(zhuǎn)染肺癌A549細(xì)胞，另于標(biāo)準(zhǔn)空白孔中加不含細(xì)胞的完全培養(yǎng)液。轉(zhuǎn)染48 h后，每孔加入20 μL MTT(2 g/L)，繼續(xù)孵育4 h后，終止培養(yǎng)。吸去培養(yǎng)液，每孔加DMSO 150 μL，在酶標(biāo)儀490 nm波長(zhǎng)處測(cè)定各孔吸光度(A)值。每組平行重復(fù)6孔，重復(fù)實(shí)驗(yàn)3次。按以下公式計(jì)算：細(xì)胞存活率(%)=A實(shí)驗(yàn)組/A對(duì)照組×100%。

2.4細(xì)胞存活數(shù)目檢測(cè)于6孔板中將miRNA-126轉(zhuǎn)染入肺癌A549細(xì)胞48 h后， 0.25%胰酶消化，用0.04%臺(tái)盼藍(lán)分別對(duì)control、negative control和miRNA-126組A549細(xì)胞進(jìn)行染色，顯微鏡下計(jì)數(shù)活細(xì)胞數(shù)(其中著色細(xì)胞為死細(xì)胞)，每個(gè)樣品計(jì)數(shù)3次。

2.5細(xì)胞集落培養(yǎng)實(shí)驗(yàn)于6孔板中轉(zhuǎn)染各組A549細(xì)胞24 h后，用0.25%胰酶消化，1 000 r/min離心5 min收集細(xì)胞，重懸細(xì)胞并計(jì)數(shù)，將細(xì)胞懸液作梯度倍數(shù)稀釋，以適當(dāng)?shù)募?xì)胞密度接種于培養(yǎng)皿中，并盡量使細(xì)胞均勻分布于板底，放入培養(yǎng)箱中連續(xù)培養(yǎng)10 d至肉眼可見(jiàn)集落時(shí)，棄原培養(yǎng)液，PBS洗滌1次后加10%甲醛固定15 min，PBS洗2次后用結(jié)晶紫染色30 min，PBS洗凈后晾干，拍照。重復(fù)實(shí)驗(yàn)3次。

2.6劃痕愈合實(shí)驗(yàn)將miRNA-126轉(zhuǎn)染肺癌A549細(xì)胞24 h后接種6孔板，待細(xì)胞鋪滿孔底，用10 μL槍頭小心在孔底劃痕，PBS清洗3次，加入含有2.5%低血清的新鮮培養(yǎng)基， 倒置顯微鏡下拍照，沿劃痕邊緣等間距取3處測(cè)量劃痕寬度，取平均值。24 h后繼續(xù)拍照，在相同觀察點(diǎn)測(cè)量劃痕寬度。劃痕愈合率(%)=(0 h劃痕寬度-24 h劃痕寬度)/0 h劃痕寬度×100%。

2.7Transwell小室實(shí)驗(yàn)將miRNA-126轉(zhuǎn)染肺癌A549細(xì)胞24 h后，消化細(xì)胞于無(wú)血清培養(yǎng)基，將含有8×107/L細(xì)胞的200 μL細(xì)胞懸液加入鋪有基質(zhì)膠的Transwell 小室上室，下室中加入600 μL含10%胎牛血清的培養(yǎng)基, 每組設(shè)置3個(gè)復(fù)孔，于37 ℃、5% CO2條件下培養(yǎng)24 h。取出Transwell小室，用棉簽擦掉Matrigel及上室未穿膜細(xì)胞，甲醇固定10 min，0.1%結(jié)晶紫染色40 min。100倍鏡下隨機(jī)選取5個(gè)視野計(jì)數(shù)穿膜細(xì)胞數(shù)目，取平均值。

2.8Western blot法檢測(cè)相關(guān)蛋白水平變化各組細(xì)胞經(jīng)RIPA裂解液(1 mL RIPA+10 μL PMSF+10 μL aprotinin)裂解30 min，冰上進(jìn)行操作。收集到EP管，BCA蛋白定量法測(cè)定蛋白濃度定量。每個(gè)泳道蛋白樣品10 μg，經(jīng)10% SDS-PAGE分離后，利用濕法將蛋白轉(zhuǎn)移至PVDF膜上，用含5% BSA的PBST封閉1 h，分別加入對(duì)應(yīng)I 抗4 ℃孵育過(guò)夜，TBST洗膜3次后，加入對(duì)應(yīng)II 抗室溫?fù)u床孵育2 h，TBST洗膜3次，ECL發(fā)光試劑盒發(fā)光顯影，定影后進(jìn)行分析。

3統(tǒng)計(jì)學(xué)處理

所有實(shí)驗(yàn)均重復(fù)3次，實(shí)驗(yàn)數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，應(yīng)用SPSS 17.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)分析。多組間比較應(yīng)用單因素方差分析，組間兩兩比較應(yīng)用Bonferroni檢驗(yàn)，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

1miRNA-126 agomir轉(zhuǎn)染肺癌A549細(xì)胞后上調(diào)miRNA-126的表達(dá)量

轉(zhuǎn)染miRNA-126 agomir可顯著提高miRNA-126在肺癌A549細(xì)胞中的表達(dá)量。miRNA-126組中miRNA-126的表達(dá)量相對(duì)陰性對(duì)照組及空白對(duì)照組明顯上調(diào)(P<0.01)，而陰性對(duì)照組和空白對(duì)照組之間差異不明顯。肺癌A549細(xì)胞經(jīng)轉(zhuǎn)染48 h后，40 nmol/L的agomir可使肺癌A549細(xì)胞的miRNA-126表達(dá)量上調(diào)約800倍，見(jiàn)圖1。

2miRNA-126對(duì)肺癌A549細(xì)胞活力的影響

MTT法檢測(cè)結(jié)果顯示，miRNA-126組的生存率與NC組和control組相比，差異有統(tǒng)計(jì)學(xué)顯著性(P<0.01)，而NC組和control組相比差異無(wú)統(tǒng)計(jì)學(xué)顯著性。結(jié)果表明，上調(diào)miRNA-126表達(dá)后肺癌A549細(xì)胞的增殖能力被顯著抑制，見(jiàn)圖2。

3miRNA-126對(duì)肺癌A549細(xì)胞增殖的影響

miRNA-126轉(zhuǎn)染入肺癌A549細(xì)胞48 h后，用0.04%臺(tái)盼藍(lán)分別對(duì)A549細(xì)胞進(jìn)行染色后，計(jì)算細(xì)胞的存活數(shù)目。miRNA-126組的細(xì)胞存活數(shù)目明顯減少，較空白對(duì)照組與陰性對(duì)照組比較差異有統(tǒng)計(jì)學(xué)顯著性(P<0.01)，見(jiàn)圖3。

Figure 1.The expression on miRNA-126 in the lung cancer A549 cells after transfection with miRNA-126 agomir. Mean±SD.n=3.**P<0.01vsNC.

圖1定量PCR方法檢測(cè)細(xì)胞轉(zhuǎn)染miRNA-126 agomir 48 h后miRNA-126表達(dá)量的上調(diào)情況

Figure 2.The cell activity of lung cancer A549 cells after transfection with miRNA-126 by MTT assay. Mean±SD.n=3.**P<0.01vsNC.

圖2MTT法檢測(cè)miRNA-126對(duì)肺癌A549細(xì)胞活力的影響

Figure 3.The number of viable A549 cells was counted by the method of Trypan blue exclusion. Mean±SD.n=3.**P<0.01vsNC.

圖3臺(tái)盼藍(lán)拒染法檢測(cè)活細(xì)胞數(shù)目

4miRNA-126對(duì)肺癌A549細(xì)胞集落形成的影響

細(xì)胞集落培養(yǎng)實(shí)驗(yàn)結(jié)果顯示，miRNA-126組集落形成數(shù)與NC組相比明顯減少(P<0.01)，細(xì)胞的增殖能力明顯受抑制，而NC組和control組集落形成數(shù)量的差異無(wú)統(tǒng)計(jì)學(xué)顯著性，見(jiàn)圖4。

Figure 4.Cell colony formation after transfection with miRNA-126. Mean±SD.n=3.**P<0.01vsNC.

圖4轉(zhuǎn)染miRNA-126后細(xì)胞集落形成情況

5miRNA-126對(duì)肺癌A549細(xì)胞遷移侵襲能力的影響

劃痕愈合實(shí)驗(yàn)及Transwell小室實(shí)驗(yàn)檢測(cè)對(duì)肺癌A549細(xì)胞遷移侵襲能力的變化。結(jié)果顯示轉(zhuǎn)染miRNA-126后，肺癌A549細(xì)胞的遷移侵襲能力受到明顯的抑制。劃痕愈合實(shí)驗(yàn)結(jié)果顯示，miRNA-126組劃痕愈合率較control組和NC組分別下降了24.76%和23.44%，但NC組和空白組之間的差異無(wú)統(tǒng)計(jì)學(xué)顯著性。miRNA-126組侵襲至Transwell小室濾膜下表面的細(xì)胞數(shù)為48.00±4.82，明顯低于control組(109.00±6.63)和NC組(107.00±5.25)，NC組和空白組之間的差異無(wú)統(tǒng)計(jì)學(xué)顯著性，見(jiàn)圖5。

6miRNA-126對(duì)EGFR/AKT/mTOR信號(hào)通路的影響

Western blot法檢測(cè)結(jié)果顯示，miRNA-126組與NC組相比， p-EGFR、p-AKT和p-mTOR水平均顯著下降(P<0.01)，NC組與對(duì)照組相比， p-EGFR、p-AKT和p-mTOR水平的差異無(wú)統(tǒng)計(jì)學(xué)顯著性，見(jiàn)圖6。

討論

MicroRNA是一類由內(nèi)源基因編碼的長(zhǎng)度約為22 個(gè)核苷酸的非編碼雙鏈RNA 分子，在細(xì)胞增殖凋亡遷移等多個(gè)生物進(jìn)程中發(fā)揮著類似癌基因或抑癌基因的作用。目前研究發(fā)現(xiàn)在多種腫瘤中表達(dá)下調(diào)，如肺癌[5]、骨肉瘤[6]、食管鱗癌[7]、肝癌[8]、胃癌[9]等腫瘤的研究中發(fā)現(xiàn)癌組織較正常組織表達(dá)下降。Liu等[10]研究甲基化測(cè)序分析發(fā)現(xiàn)miRNA-126位于EGFL7基因的內(nèi)含子中，EGFL7基因啟動(dòng)子的高甲基化導(dǎo)致miRNA-126的表達(dá)下調(diào)，過(guò)表達(dá)miRNA-126或抑制ADAM9的表達(dá)可下調(diào)EGFR/AKT信號(hào)活化，抑制食管磷癌細(xì)胞的體內(nèi)外生長(zhǎng)及遷移，該研究說(shuō)明miRNA-126在腫瘤表達(dá)下調(diào)為表觀遺傳學(xué)機(jī)制所致。我們?cè)谇捌谘芯恐欣胢iRNA基因芯片技術(shù)也發(fā)現(xiàn)miRNA-126在肺癌組織與癌旁組織中差異表達(dá)，我們推測(cè)其在非小細(xì)胞肺癌組織中表達(dá)下調(diào)極可能也是由表觀遺傳學(xué)機(jī)制所致。

目前有關(guān)miRNA-126在肺癌中的研究較少，如Edit等[11]認(rèn)為miRNA-126靶向作用于SLC7A5，延遲H69細(xì)胞G1期，從而抑制細(xì)胞增殖。Crawford等[12]的研究報(bào)道m(xù)iRNA-126可能通過(guò)介導(dǎo)Crk蛋白的表達(dá)而抑制肺癌細(xì)胞的侵襲和遷移。Liu等[13]研究認(rèn)為miRNA-126通過(guò)作用于VEGF表達(dá)抑制肺癌細(xì)胞增殖。本研究通過(guò)轉(zhuǎn)染miRNA-126使肺癌細(xì)胞中miRNA-126的表達(dá)量增加探討其對(duì)肺癌A549細(xì)胞增殖、遷移、侵襲等生物學(xué)行為的變化，結(jié)果顯示過(guò)表達(dá)miRNA-126能顯著抑制肺癌A549細(xì)胞的增殖、遷移和侵襲。

Figure 5.The effect of miRNA-126 on A549 cell migration and invasion. A: the migration ability of A549 cells transfected with miRNA-126 was examined by wound healing assay; B: the invasion ability of A549 cells transfected with miRNA-126 was measured by Transwell assay. Mean±SD.n=3.**P<0.01vsNC.

圖5miRNA-126對(duì)肺癌A549細(xì)胞遷移和侵襲能力的影響

Figure 6.The protein levels of p-EGFR, p-AKT and p-mTOR in the lung cancer A549 cells transfected with miRNA-126. Mean±SD.n=3.**P<0.01vsNC.

圖6轉(zhuǎn)染miRNA-126對(duì)肺癌A549細(xì)胞EGFR/AKT/mTOR通路相關(guān)蛋白水平的影響

EGFR是表皮生長(zhǎng)因子(epidermal growth factor，EGF)細(xì)胞增殖和信號(hào)傳導(dǎo)的受體。在多種惡性腫瘤中，EGFR發(fā)揮了重要作用，包括促進(jìn)增殖、減少凋亡、提高腫瘤細(xì)胞運(yùn)動(dòng)能力和促進(jìn)血管新生[14-15]等。EGFR突變導(dǎo)致其表達(dá)或活性異常，進(jìn)而會(huì)促進(jìn)腫瘤的發(fā)生。EGFR下游信號(hào)主要包括 PI3K/AKT、Ras/Raf/MEK/MAPK、STAT3等通路。Schettino等[16]研究認(rèn)為 EGFR 在肺癌中過(guò)表達(dá)的發(fā)生率為37%～60%，并且臨床分期越晚，EGFR的表達(dá)越強(qiáng)，說(shuō)明EGFR 的表達(dá)與肺癌的發(fā)生發(fā)展密切相關(guān)。然而，目前miRNA-126在肺癌中有關(guān)EGFR的信號(hào)通路研究尚不清楚。本實(shí)驗(yàn)通過(guò)分析miRNA-126與EGFR信號(hào)通路分子間的相互關(guān)系，進(jìn)一步明確miR-126在肺癌中的作用機(jī)制。研究結(jié)果顯示miRNA-126可以顯著降低肺癌A549細(xì)胞中p-EGFR、p-AKT和p-mTOR蛋白水平。說(shuō)明miRNA-126可能通過(guò)下調(diào)EGFR/AKT/mTOR信號(hào)通路影響肺癌A549細(xì)胞的增殖、遷移和侵襲。

綜上所述，本實(shí)驗(yàn)通過(guò)細(xì)胞轉(zhuǎn)染使肺癌A549細(xì)胞miRNA-126過(guò)表達(dá)，分析肺癌細(xì)胞的增殖、遷移、侵襲等生物學(xué)行為變化，并探討引起這一變化的可能作用機(jī)制。實(shí)驗(yàn)結(jié)果表明miRNA-126能夠抑制肺癌A549細(xì)胞的增殖、遷移和侵襲，同時(shí)抑制了EGFR/AKT/mTOR信號(hào)通路。我們還將繼續(xù)研究miRNA-126的靶基因，通過(guò)基因敲除或過(guò)表達(dá)該靶基因，進(jìn)而構(gòu)建穩(wěn)定的細(xì)胞株，分析EGFR/AKT/mTOR信號(hào)通路與miRNA-126的靶基因之間的相互作用，將為miRNA-126應(yīng)用于肺癌的臨床治療提供實(shí)驗(yàn)依據(jù)。

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(責(zé)任編輯： 陳妙玲， 羅森)

miRNA-126 inhibits proliferation, migration and invasion of human lung cancer A549 cells via EGFR/AKT/mTOR pathway

TANG Xia-li, JIAO De-min, CHEN Jun, WANG Jian, CHEN Qing-yong

(DepartmentofRespiratoryMedicine,The117thHospitalofPLA,Hangzhou310004,China.E-mail:cqy117@163.com)

[ABSTRACT]AIM: To investigate the effects of microRNA (miRNA)-126 on the proliferation, migration and invasion of human lung cancer cell lines, and to explore its mechanism. METHODS: The A549 cells were transfected with miRNA-126 agomir by Lipofectamine 2000. The expression of miRNA-126 was detected by real-time PCR. The cell activity was detected by MTT assay. The number of viable A549 cells was counted by the method of Trypan blue exclusion. The cell colony-forming capability was determined by cell colony formation test. The cell migration and invasion abilities were assayed by wound healing and Transwell methods, respectively. The protein levels of p-EGFR, EGFR, p-AKT, AKT, p-mTOR and mTOR were determined by Western blot. RESULTS: The expression level of miRNA-126 was significantly increased in the A549 cells compared with negative control (NC) group and control group (P<0.01). The proliferation of A549 cells was decreased extremely after transfected with the miRNA-126 agomir (P<0.01), so did the result of the cell colony-formation test. The migration and invasion abilities of the lung cancer cells were also significantly inhibited. The protein levels of p-EGFR, p-AKT and p-mTOR were significantly down-regulated compared with NC group and control group (P<0.01). CONCLUSION: Over-expression of miRNA-126 significantly inhibits the proliferation, migration and invasion ability of human lung cancer A549 cells by down-regulation of EGFR/AKT/mTOR pathway．

[KEY WORDS]MicroRNA-126; EGFR/AKT/mTOR pathway; Lung cancer; Cell proliferation

doi:10.3969/j.issn.1000- 4718.2016.03.012

[中圖分類號(hào)]R730.23

[文獻(xiàn)標(biāo)志碼]A

通訊作者△Tel: 0571-28084872; E-mail: cqy117@163.com

*[基金項(xiàng)目]浙江省公益性技術(shù)應(yīng)用研究計(jì)劃(No.2013C33209;No.2014C33277)；杭州市醫(yī)療衛(wèi)生科研項(xiàng)目(No.20130633B29;No.20140633B40)

[收稿日期]2015- 09- 22[修回日期] 2015- 12- 03

[文章編號(hào)]1000- 4718(2016)03- 0458- 06

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