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(1.廣東省第二人民醫(yī)院呼吸內(nèi)科，廣東 廣州 510317；2.廣東省第二人民醫(yī)院心胸外科)

·基礎(chǔ)醫(yī)學(xué)·

AEG-1抑制miR-137對(duì)非小細(xì)胞肺癌增殖調(diào)控作用

常永梅1*，王明智2，孫聰1，顏文森1，劉慶峰1，江小運(yùn)1，王鈞1

(1.廣東省第二人民醫(yī)院呼吸內(nèi)科，廣東 廣州 510317；2.廣東省第二人民醫(yī)院心胸外科)

目的探討AEG-1在miR-137抑制非小細(xì)胞肺癌(NSCLC)增殖中的作用及機(jī)制。方法收集2013年2月～2014年5月在本院手術(shù)治療的65例NSCLC患者腫瘤標(biāo)本，提取RNA，qRT-PCR檢測(cè)NSCLC組織、A549、H460及16HBE細(xì)胞中miR-137與AEG-1的表達(dá)，統(tǒng)計(jì)分析NSCLC組織中miR-137與膠質(zhì)細(xì)胞上調(diào)基因1(AEG-1)表達(dá)的相關(guān)性；將A549細(xì)胞根據(jù)不同轉(zhuǎn)染試劑分成4組：轉(zhuǎn)染miR-137 mimics及空載體組，轉(zhuǎn)染miR-137 mimics及AEG-1表達(dá)載體組，轉(zhuǎn)染miR-137 inhibitor及siRNA無(wú)關(guān)序列組，轉(zhuǎn)染miR-137 inhibitor及AEG-1 siRNA組。用Lipofectamine 2000進(jìn)行轉(zhuǎn)染，生長(zhǎng)曲線實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后細(xì)胞增殖情況。結(jié)果在NSCLC組織中miR-137與AEG-1的表達(dá)呈負(fù)相關(guān)；miR-137表達(dá)高的NSCLC細(xì)胞中AEG-1表達(dá)降低；共同轉(zhuǎn)染miR-137 mimics與AEG-1過(guò)表達(dá)載體的A549細(xì)胞在48 h的細(xì)胞數(shù)高于對(duì)照組，而共同轉(zhuǎn)染miR-137 inhibitor與AEG-1 siRNA的A549細(xì)胞在48 h、72 h的細(xì)胞數(shù)低于對(duì)照組。結(jié)論miR-137與AEG-1在NSCLC組織及細(xì)胞中表達(dá)呈負(fù)相關(guān)；AEG-1逆轉(zhuǎn)miR-137對(duì)NSCLC細(xì)胞增殖的作用。

miR-137；非小細(xì)胞肺癌；膠質(zhì)細(xì)胞上調(diào)基因1；增殖

肺癌是全球引起死亡最高的惡性腫瘤。大約70%～80%的肺癌是非小細(xì)胞肺癌(non samll cell lung cancer，NSCLC)，包括鱗狀細(xì)胞癌、腺癌、大細(xì)胞癌[1]。非小細(xì)胞肺癌的預(yù)后很差，盡管今年化療藥物研究進(jìn)展及治療手段的進(jìn)步，NSCLC的5年總體生存率仍不超過(guò)11%。NSCLC的主要死亡原因是化療耐藥性和轉(zhuǎn)移，但是NSCLC轉(zhuǎn)移的機(jī)制仍不十分清楚[2]。

微小RNA是小的非編碼RNA，其在腫瘤發(fā)生和發(fā)展中發(fā)揮重要作用。miRNAs與靶基因的3’非翻譯區(qū)(3’ untranslation regions，3’-UTRs)結(jié)合，引起靶基因mRNA降解或抑制翻譯[3]。越來(lái)越多的證據(jù)表明miRNAs在NSCLC的轉(zhuǎn)移過(guò)程中發(fā)揮重要作用。許多miRNA在NSCLC中發(fā)揮抑瘤作用，抑制轉(zhuǎn)移，如：miR-200c[4]、miR-26b[5]，而另一些miRNA發(fā)揮促進(jìn)NSCLC轉(zhuǎn)移的功能，如：miR-1260b[6]。miR-137通過(guò)靶向SLC22A18、Cdc42、Cdk6抑制NSCLC增殖[7]，也可通過(guò)靶向BMP7抑制NSCLC細(xì)胞侵襲轉(zhuǎn)移[8]。有研究表明，miR-137通過(guò)靶向膠質(zhì)細(xì)胞上調(diào)基因(Astrocyte elevated gene 1，AEG-1)抑制卵巢癌細(xì)胞生長(zhǎng)[9]。而NSCLC中，miR-137與AEG-1的表達(dá)調(diào)控關(guān)系尚無(wú)報(bào)道。本研究擬通過(guò)研究miR-137在NSCLC中對(duì)AEG-1表達(dá)調(diào)控的作用，探討miR-137抑制NSCLC侵襲轉(zhuǎn)移的機(jī)制。

1 材料與方法

1.1細(xì)胞培養(yǎng)NSCLC細(xì)胞株A549、H460及正常的肺支氣管上皮細(xì)胞系16HBE從ATCC購(gòu)買。細(xì)胞培養(yǎng)基：DMEM，10%胎牛血清，2 μmol/L谷氨酰胺，100 IU/mL 青霉素及100 μg/mL 硫酸鏈霉素，在37 ℃ 5%二氧化碳環(huán)境中培養(yǎng)。

1.2臨床標(biāo)本收集及資料人非小細(xì)胞肺癌取自廣東省第二人民醫(yī)院2013年2月～2014年5月手術(shù)治療的65例患者，這些患者中無(wú)人接受手術(shù)前化療。記錄患者年齡、性別、組織學(xué)分級(jí)、腫瘤大小、浸潤(rùn)深度和淋巴結(jié)轉(zhuǎn)移等資料。本研究經(jīng)廣東省第二醫(yī)學(xué)倫理委員會(huì)批準(zhǔn)。

1.3試劑AEG-1過(guò)表達(dá)載體購(gòu)自武漢三鷹公司。AEG-1 siRNA購(gòu)自Qiagen公司。轉(zhuǎn)染試劑為L(zhǎng)ipofectamine 2000(Life)。

1.4總RNA提取、逆轉(zhuǎn)錄用Trizol(Life)提取RNA。用Nanodrop 2000(Thermo)測(cè)定RNA濃度及純度。用cDNA合成試劑盒及miRNA特異性檢測(cè)試劑盒(Genecopeoia)。cDNA合成的條件為：50 ℃ 30 min，85 ℃ 5 min。

1.5細(xì)胞轉(zhuǎn)染將NSCLC A549細(xì)胞接種于6孔板，分成4組：轉(zhuǎn)染miR-137 mimics及空載體組，轉(zhuǎn)染miR-137 mimics及AEG-1表達(dá)載體組，轉(zhuǎn)染miR-137 inhibitor及siRNA無(wú)關(guān)序列組，轉(zhuǎn)染miR-137 inhibitor及AEG-1 siRNA組。用Lipofectamine 2000進(jìn)行轉(zhuǎn)染，根據(jù)轉(zhuǎn)染試劑說(shuō)明書進(jìn)行操作。轉(zhuǎn)染48 h后提取RNA及蛋白。

1.6生長(zhǎng)曲線將A549細(xì)胞接種至24孔板，5 000/孔。細(xì)胞貼壁后，分成4組(同1.5分組及名稱)進(jìn)行處理于轉(zhuǎn)染24 h、48 h、72 h行細(xì)胞計(jì)數(shù)，繪制生長(zhǎng)曲線。

1.7熒光實(shí)時(shí)定量PCR(quantitative real-time PCR，qRT-PCR) 以2-ΔΔCT表示基因或miRNA表達(dá)，ΔΔCT = (CTmiRNA-CTU6)癌- (CTmiRNA-CTU6)癌旁。AEG-1的表達(dá)以GAPDH為內(nèi)參，使用以下引物:F:GAAGGTGAAGGTCGGAGTC,R:GAAGATGGTGATGGGATTTC。ΔΔCT= (CTAEG-1-CTGAPDH)癌-(CTAEG-1-CTGAPDH)癌旁，儀器為L(zhǎng)ight Cycler 480 II(Roche Diagnostics;Indianapolis,IN)。

1.8統(tǒng)計(jì)學(xué)方法采用SPSS13.0統(tǒng)計(jì)軟件對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行分析，兩組間均數(shù)比較采用t檢驗(yàn)，多組間均數(shù)比較采用方差分析，相關(guān)性分析用Pearson檢驗(yàn)，以P<0.05為差異有顯著性。

2 結(jié) 果

2.1 miR-137與AEG-1在NSCLC組織及細(xì)胞中表達(dá)呈負(fù)相關(guān)qRT-PCR檢測(cè)了NSCLC組織及細(xì)胞中miR-137與AEG-1的表達(dá)。相關(guān)性分析顯示在NSCLC組織中miR-137與AEG-1的表達(dá)呈負(fù)相關(guān)(r=-0.5285，P<0.001)(圖1)。miR-137在NSCLC細(xì)胞中的表達(dá)顯著低于肺支氣管上皮細(xì)胞16HBE(P<0.05)，而NSCLC中AEG-1的表達(dá)明顯高于16HBE(P<0.01)，miR-137及AEG-1在NSCLC及16HBE細(xì)胞中的表達(dá)相反(圖2)。

2.2 AEG-1逆轉(zhuǎn)miR-137對(duì)NSCLC細(xì)胞增殖的作用在轉(zhuǎn)染miR-137 mimics的A549細(xì)胞中同時(shí)轉(zhuǎn)染AEG-1過(guò)表達(dá)載體，細(xì)胞48 h細(xì)胞數(shù)高于對(duì)照組(P<0.05)；在轉(zhuǎn)染miR-137 inhibitor的A549細(xì)胞中同時(shí)轉(zhuǎn)染AEG-1 siRNA，細(xì)胞48 h及72 h的細(xì)胞數(shù)低于對(duì)照組(P<0.05。見(jiàn)圖3。

圖1 NSCLC組織中miR-137與AEG-1表達(dá)相關(guān)性分析

圖2 miR-137及AEG-1在NSCLC細(xì)胞及肺支氣管上皮細(xì)胞中的表達(dá) 與16 HBE比較，*P<0.05，**P<0.01

3 討 論

研究證實(shí)，miRNAs 在NSCLC發(fā)生發(fā)展過(guò)程中發(fā)揮重要作用[10]。miRNAs作為腫瘤抑制或致癌基因參與腫瘤發(fā)展的多個(gè)過(guò)程，包括細(xì)胞增殖、凋亡、遷移和侵襲[11]。miR-137在多種腫瘤中發(fā)揮抑瘤作用。最新的研究表明，miR-137可通過(guò)靶向表皮生長(zhǎng)因子受體抑制甲狀腺癌的增殖[12]。在肺癌中，miR-137可抑制肺癌的增殖并促進(jìn)肺癌的紫杉醇和順鉑的藥物敏感性[13]， 還能通過(guò)靶向骨形態(tài)發(fā)生蛋白(bone morphogenetic protein 7，BMP7)抑制NSCLC細(xì)胞遷移和增殖[8]，也可通過(guò)抑制KIT的表達(dá)抑制小細(xì)胞肺癌細(xì)胞增殖[14]。

AEG-1已被確認(rèn)為是在許多惡性腫瘤進(jìn)展和轉(zhuǎn)移過(guò)程中的一個(gè)重要的致癌基因，它可調(diào)控Ras、PI3K / Akt、NF-κB、ERK和Aurora-A激酶等多個(gè)信號(hào)通路。AEG-1在肺癌中表達(dá)增加，促進(jìn)NSCLC遷移[15]，可通過(guò)活化Wnt/β-catenin信號(hào)通路誘導(dǎo)肺癌細(xì)胞發(fā)生上皮間質(zhì)樣變[16]。針對(duì)AEG-1的治療手段應(yīng)具有抑制轉(zhuǎn)移、腫瘤生長(zhǎng)、提高化療敏感性等特性。研究表明，AEG-1在腫瘤中的表達(dá)受多個(gè)miRNA調(diào)控，其中包括miR-137[9]。但miR-137與AEG-1在肺癌中的表達(dá)尚無(wú)研究。因此，本研究通過(guò)qRT-PCR的方法檢測(cè)了65例NSCLC組織中miR-137及AEG-1 mRNA的表達(dá)，并且證實(shí)兩者在NSCLC組織中表達(dá)呈反比。這與已報(bào)道的兩者在卵巢癌中的表達(dá)變化相似。已知，miR-137為抑癌miRNA，在肺癌可抑制細(xì)胞增殖，而AEG-1在肺癌中表達(dá)增加，并且參與促進(jìn)NSCLC細(xì)胞增殖[17]。作為miR-137的靶基因，AEG-1是否參與了miR-137對(duì)NSCLC細(xì)胞功能的調(diào)控？于是，本研究檢測(cè)了miR-137與AEG-1共同對(duì)NSCLC細(xì)胞增殖的影響。結(jié)果證實(shí)，miR-137與AEG-1共同高表達(dá)組細(xì)胞增殖率明顯高于miR-137高表達(dá)組，miR-137與AEG-1共同表達(dá)抑制組細(xì)胞增殖率低于miR-137高表達(dá)組。這說(shuō)明AEG-1作為miR-137的靶基因逆轉(zhuǎn)了miR-137抑制NSCLC細(xì)胞增殖的作用。

圖3 miR-137與AEG-1對(duì)NSCLC細(xì)胞增殖的影響 A：miR-137 mimics及AEG-1對(duì)A549細(xì)胞增殖的影響；B：miR-137 inhibitor及AEG-1 siRNA對(duì)A549細(xì)胞增殖的影響。Scramble：無(wú)關(guān)序列；NC：inhibitor的對(duì)照序列； *P<0.05

本研究證實(shí)了NSCLC組織及細(xì)胞中miR-137與AEG-1表達(dá)呈負(fù)相關(guān)，并且AEG-1可逆轉(zhuǎn)miR-137對(duì)NSCLC細(xì)胞增殖抑制作用。但是AEG-1的這種作用調(diào)控的下游信號(hào)通路尚不清楚，這成為我們后續(xù)研究的方向。

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AEG-1ReversestheInhibitionofNon-smallCellLungCancerProliferationInducedbyMiR-137

CHANG Yongmei,WANG Mingzhi,SUN Cong,et al

(DepartmentofRespiratoryMedicine,GuangdongNo.2ProvincialPeopleHospital，Guangzhou,Guangdong510317,China)

ObjectiveTo investigate the the function of AEG-1 (astrocyte elevated ) in the process of miR-137 inhibiting the proliferation of non-small cell lung cancer (NSCLC).MethodsSixty five samples in our hospital from Feb.2013 to May 2014 were collected.And RNA was extracted from these samples.QRT-PCR was conducted to detect the expression of miR-137 and AEG-1 in NSCLC tissues,and A549,H460 and 16HBE cells.The correlation between miR-137 and AEG-1 in NSCLC tissues was statistically analyzed.A549 cells were divided into four groups according to the transfection treatment:miR-137 mimics plus empty vector group,miR-137 mimics plus AEG-1 expression vector group,miR-137 inhibitor plus scramble sequence group,and miR-137 inhibitor plus AEG-1 siRNA group.The growth curve assay was performed to detect the cells proliferation after transfection treatment.ResultThe expression of miR-137 is negatively correlated with the AEG-1 in NSCLC tissues.The expression of AEG-1 was decreased in miR-137 overexpressed NSCLC cells.The cell number of A549 co-transfected with miR-137 mimics and AEG-1 expression vector was larger than the control group in 48 h.And,the cell number of A549 co-transfected with miR-137 inhibitor and AEG-1 siRNA was smaller than the control group in 48 h and 72 h.ConclusionThe expression of miR-137 is negatively correlated with the expression of AEG-1 in NSCLC tissues and cells;AEG-1 reversed the effect of miR-137 in the proliferation of NSCLC cell.

miR-137; non-small cell lung cancer; AEG-1; proliferation

10.15972/j.cnki.43-1509/r.2016.03.010

2016-01-28；

2016-04-28

廣東省科技計(jì)劃項(xiàng)目(2014A020212447).

*通訊作者，E-mail:cym891@163.com.

R734.2

A

蔣湘蓮)