羅衛(wèi)民，張軍，余宗濤，林稱意，郭家龍

(湖北醫(yī)藥學(xué)院附屬十堰市太和醫(yī)院心胸外科1、檢驗(yàn)科2，湖北 十堰 442000)

VEGF逆轉(zhuǎn)miR-200b促進(jìn)非小細(xì)胞肺癌增殖的作用

羅衛(wèi)民1，張軍1，余宗濤2，林稱意1，郭家龍1

(湖北醫(yī)藥學(xué)院附屬十堰市太和醫(yī)院心胸外科1、檢驗(yàn)科2，湖北 十堰 442000)

目的探討血管內(nèi)皮生長(zhǎng)因子(VEGF)在miR-200b抑制非小細(xì)胞肺癌(NSCLC)增殖中的作用及機(jī)制。方法選擇2013年2月至2014年5月在本院經(jīng)手術(shù)治療的65例NSCLC患者的腫瘤組織，qRT-PCR檢測(cè)NSCLC組織中miR-200b與VEGF的表達(dá)，并統(tǒng)計(jì)分析NSCLC組織中miR-200b與VEGF表達(dá)的相關(guān)性；常規(guī)培養(yǎng)A549、H460及16HBE細(xì)胞，qRT-PCR檢測(cè)各組細(xì)胞中miR-200b與VEGF的表達(dá)；將常規(guī)培養(yǎng)的A549細(xì)胞隨機(jī)分為四組，即miR-200b mimics+vector、miR-200b mimics+VEGF組、miR-200b inhibitor+scramble組、miR-200b inhibitor+VEGF-siRNA組，其中miR-200b mimics+vector組與miR-200b inhibitor+scramble組作為陰性對(duì)照，采用MTT法檢測(cè)各組吸光度值(OD值)。結(jié)果qRT-PCR結(jié)果顯示，miR-200b在NSCLC組織的表達(dá)水平為(0.682± 0.106)，VEGF在NSCLC組織的表達(dá)水平為(2.731±0.597)，相關(guān)性分析顯示在NSCLC組織中miR-200b與VEGF的表達(dá)呈負(fù)相關(guān)(r=-0.514，P＜0.001)；miR-200b在A549、H460細(xì)胞中的表達(dá)顯著低于16HBE細(xì)胞，差異有統(tǒng)計(jì)學(xué)意義(P=0.000)，而VEGF在A549、H460細(xì)胞中的表達(dá)明顯高于16HBE細(xì)胞，差異有統(tǒng)計(jì)學(xué)意義（P=0.000）；MTT結(jié)果顯示，miR-200b mimics+VEGF組A549細(xì)胞在48 h、72 h的細(xì)胞增殖率明顯高于miR-200b mimics+vector組，miR-200b inhibitor+VEGF-siRNA組A549細(xì)胞在48 h、72 h的細(xì)胞增殖率明顯低于miR-200b inhibitor+scramble組，差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05)。結(jié)論miR-200b與VEGF在NSCLC組織及細(xì)胞中表達(dá)呈負(fù)相關(guān)；VEGF逆轉(zhuǎn)miR-200b對(duì)NSCLC細(xì)胞增殖的作用。

miR-200b；非小細(xì)胞肺癌；血管內(nèi)皮生長(zhǎng)因子；增殖

肺癌是全球死亡率最高的惡性腫瘤，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)展及治療手段有所進(jìn)步，NSCLC的5年總體生存率仍不超過(guò)11%。其主要死亡原因是化療耐藥性和轉(zhuǎn)移，但NSCLC轉(zhuǎn)移的機(jī)制仍不十分清楚[2]。

微小RNA(miRNAs)是一組包含19～25個(gè)核苷酸的非編碼小分子單鏈RNA，是基因表達(dá)的一個(gè)重要調(diào)控因子。通過(guò)與靶基因3′-UTR(3′-非編碼區(qū))形成完全或不完全地堿基配對(duì)，miRNA可導(dǎo)致靶mRNA的翻譯抑制或降解，并在轉(zhuǎn)錄后水平上調(diào)控靶基因表達(dá)。miRNA同時(shí)也參與多種細(xì)胞生物學(xué)行為，包括細(xì)胞的分化、增殖、遷移、代謝及凋亡等過(guò)程。過(guò)去的研究已經(jīng)證明miRNA表達(dá)譜在不同的腫瘤中的作用，同時(shí)也表明了miRNA的表達(dá)異常與腫瘤的發(fā)生發(fā)展息息相關(guān)[3-4]。據(jù)報(bào)道，目前已有一些研究證實(shí)miRNA在NSCLC中能發(fā)揮抗腫瘤作用，如miR-145[5]，而還有許多miRNA能在NSCLC中促進(jìn)腫瘤的轉(zhuǎn)移，如miR-141[6]，miRNA-197[7]。miR-200b能抑制支氣管上皮細(xì)胞的上皮間葉轉(zhuǎn)化(EMT)及腫瘤細(xì)胞轉(zhuǎn)移[8]，同時(shí)miR-200b還能下調(diào)E盒結(jié)合鋅指蛋白2(ZEB2)及E2F3的表達(dá)，增加E-鈣連接素表達(dá)，從而抑制耐藥性NSCLC細(xì)胞增殖，并促進(jìn)腫瘤細(xì)胞凋亡[9-10]。

血管內(nèi)皮生長(zhǎng)因子(VEGF)是一種重要的血管生成促成因子，通過(guò)與特異性受體結(jié)合從而刺激血管內(nèi)皮細(xì)胞增殖，增加血管通透性，誘導(dǎo)腫瘤新生血管及淋巴管形成，其過(guò)度表達(dá)與腫瘤的形成、發(fā)展、轉(zhuǎn)移等病理過(guò)程密切相關(guān)[11]。研究表明VEGF在多種實(shí)體腫瘤中高表達(dá)，如胃癌[3]、肝癌[12]、乳腺癌[13]、NSCLC[14]；Fossella等[14]研究表明在NSCLCⅠ～Ⅳ臨床分期中，臨床分期越高患者血清中VEGF水平也升高。此外，miR-200b通過(guò)靶向作用于VEGF及其受體負(fù)性調(diào)控VEGF信號(hào)通路，從而抑制腫瘤細(xì)胞血管的生成[15]。而NSCLC中，miR-200b與VEGF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-200b在NSCLC中對(duì)VEGF表達(dá)調(diào)控的作用，探討miR-200b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)胞系人支氣管上皮細(xì)胞(16HBE)，從美國(guó)標(biāo)準(zhǔn)生物品收藏中心(ATCC)購(gòu)買。細(xì)胞培養(yǎng)基：DMEM，10%胎牛血清，2μmol/L谷氨酰胺，100 IU/mL青霉素及100μg/mL硫酸鏈霉素，在37℃、5%二氧化碳環(huán)境培養(yǎng)。

1.2 臨床標(biāo)本收集及資料 人非小細(xì)胞肺癌取自本院2013年2月至2014年5月手術(shù)治療的65例患者，所有患者均無(wú)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)醫(yī)學(xué)倫理委員會(huì)批準(zhǔn)。

1.3 試劑 miR-200b mimics、miR-200b inhibitor、VEGF過(guò)表達(dá)載體、vector、VEGF siRNA、scramble購(gòu)自Genecopoeia公司。轉(zhuǎn)染試劑為L(zhǎng)ipofectamine 2000，購(gòu)自invitrogen公司。

1.4 總RNA提取、逆轉(zhuǎn)錄 用Trizol(invitrogen)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)胞分組與轉(zhuǎn)染 將NSCLCA549細(xì)胞分為miR-200b mimics+vector組(共轉(zhuǎn)染miR-200b mimics與vector)、miR-200b mimics+VEGF組(共轉(zhuǎn)染miR-200b mimics與VEGF)、miR-200b inhibitor+scramble組(共轉(zhuǎn)染miR-200b inhibitor與scramble)、miR-200b inhibitor+VEGF-siRNA組(共轉(zhuǎn)染miR-200b inhibitor與VEGF-siRNA)，其中miR-200b mimics+vector組與miR-200b inhibitor+scramble組作為陰性對(duì)照。將A549細(xì)胞接種于6孔板，將上述轉(zhuǎn)染用的質(zhì)粒分別轉(zhuǎn)染細(xì)胞。根據(jù)轉(zhuǎn)染試劑說(shuō)明書進(jìn)行操作。

1.6 MTT實(shí)驗(yàn) 將上述轉(zhuǎn)染48 h后的4組A549細(xì)胞接種于96孔板中，每組設(shè)5個(gè)復(fù)孔，分別培養(yǎng)24 h、48 h、72 h至臨近90%飽和度，每孔加滅菌MTT液(5 mg/mL)20 μL，孵育4 h后取出，每孔中加入DMSO 150 μL，低速振蕩10 min，選擇波長(zhǎng)為570 nm，在酶標(biāo)儀上測(cè)定各孔吸光值，記錄結(jié)果，實(shí)驗(yàn)重復(fù)3次。

1.7 熒光實(shí)時(shí)定量PCR(quantitative real-time PCR，qRT-PCR)PCR擴(kuò)增反應(yīng)體系為20 μL，其中包括MiR-PCR primers(5 mmol/L)0.4 μL，miRNA RT product 2.0 μL，Taq DNA polymerase 5 U/μL)0.2 μL，2×SYBR Mix 10 μL，滅菌蒸餾水7.4 μL。循環(huán)體系為：95℃，3 min；95℃，12 s；62℃，35 s；72℃，30 s，一共40個(gè)循環(huán)。以β-actin為內(nèi)參，所測(cè)定的miR-200b的相對(duì)表達(dá)量采用2-ΔΔCT法分析。

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，計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(±s)表示，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為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 miR-200b與VEGF在NSCLC組織及細(xì)胞中的表達(dá) qRT-PCR檢測(cè)了NSCLC組織及細(xì)胞中miR-200b與VEGF的表達(dá)。結(jié)果顯示，miR-200b在NSCLC組織的表達(dá)水平為(0.682±0.106)，VEGF在NSCLC組織的表達(dá)水平為(2.731±0.597)，相關(guān)性分析顯示在NSCLC組織中miR-200b與VEGF的表達(dá)呈負(fù)相關(guān)(r=-0.514，P＜0.001)。miR-200b在NSCLC細(xì)胞中的表達(dá)顯著低于16HBE，差異有統(tǒng)計(jì)學(xué)意義(F= 214.670，P=0.000)，而NSCLC中VEGF的表達(dá)明顯高于16HBE，差異有統(tǒng)計(jì)學(xué)意義(F=107.820，P=0.000)，miR-200b及VEGF在NSCLC及16HBE細(xì)胞中的表達(dá)相反，見表1。

表1 miR-200b及VEGF在NSCLC細(xì)胞及肺支氣管上皮細(xì)胞中表達(dá)(±s)

表1 miR-200b及VEGF在NSCLC細(xì)胞及肺支氣管上皮細(xì)胞中表達(dá)(±s)

注：與16HBE細(xì)胞相比較，aP＜0.01。

16HBE細(xì)胞A549細(xì)胞2.158±0.573 0.740±0.092 0.536±0.084a2.905±0.746a

2.2 VEGF逆轉(zhuǎn)miR-200b對(duì)NSCLC細(xì)胞增殖的作用 MTT結(jié)果顯示，在轉(zhuǎn)染miR-200b mimics+ VEGF組的A549細(xì)胞48 h與72 h的OD值高于miR-200b mimics+vector組，差異有統(tǒng)計(jì)學(xué)意義；在轉(zhuǎn)染miR-200b inhibitor+VEGF-siRNA組的A549細(xì)胞48 h與72 h的OD值低于miR-200b inhibitor+scramble，差異有統(tǒng)計(jì)學(xué)意義(48 h：miR-200b mimics+VEGF組t=4.695，P=0.009；miR-200b inhibitor+VEGF-siRNA組t=9.035，P=0.001；72 h：miR-200b mimics+VEGF組t=5.266，P=0.006；miR-200b inhibitor+VEGF-siRNA組t=7.046，P=0.002)。

表2 miR-200b與VEGF對(duì)NSCLC細(xì)胞增殖的影響(±s)

表2 miR-200b與VEGF對(duì)NSCLC細(xì)胞增殖的影響(±s)

注：與miR-200b mimics+vector組或miR-200b inhibitor+scramble組相比較，aP＜0.05，bP＜0.01。

時(shí)間24 h 48 h 72 h miR-200b inhibitor+VEGF-siRNA 0.413±0.008 0.371±0.010a0.405±0.016bmiR-200b mimics+vector 0.428±0.007 0.472±0.015 0.561±0.014 miR-200b mimics+VEGF 0.436±0.010 0.590±0.017a0.745±0.021bmiR-200b inhibitor+scramble 0.421±0.011 0.486±0.012 0.594±0.018

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[16]。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[17]。miR-200b在多種腫瘤中發(fā)揮抑瘤作用。最近有研究表明，miR-200b能靶向調(diào)節(jié)Bmi-1癌基因抑制舌癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化[18]，還能增強(qiáng)前列腺癌細(xì)胞對(duì)化療藥物的敏感性，抑制其增殖及轉(zhuǎn)移[19]。此外，miR-200b通過(guò)靶向Ras鳥苷酸結(jié)合蛋白超家族的新成員(RND3)來(lái)調(diào)節(jié)細(xì)胞周期蛋白D1(cyclin D1)表達(dá)水平從而促進(jìn)宮頸癌細(xì)胞周期停滯[20]。在肺癌中，miR-200b通過(guò)靶向作用轉(zhuǎn)錄調(diào)控因子E2F3逆轉(zhuǎn)肺癌對(duì)多西他賽化療耐藥[9]，還能通過(guò)靶向血管內(nèi)皮生長(zhǎng)因子受體1(FLT1/VEGFR1)抑制肺癌細(xì)胞遷移和增殖[21]。

VEGF是一種重要的血管生成調(diào)節(jié)因子，主要由腫瘤細(xì)胞分泌。人VEGF存在5種同型異構(gòu)體，其中VEGF165是生物活性最強(qiáng)的分子類型，而VEGF已被看作是許多不同惡性腫瘤生長(zhǎng)和擴(kuò)散轉(zhuǎn)移過(guò)程中的一個(gè)關(guān)鍵的致癌基因[22]。早期的研究發(fā)現(xiàn)，VEGF參與肺癌浸潤(rùn)與轉(zhuǎn)移機(jī)制存在兩種形式，一種方式可能通過(guò)與內(nèi)皮細(xì)胞上的VEGFR結(jié)合，經(jīng)過(guò)旁分泌形式促進(jìn)腫瘤新生血管形成及增加血管通透性從而導(dǎo)致肺癌細(xì)胞浸潤(rùn)與轉(zhuǎn)移；另一種方式可能通過(guò)自分泌機(jī)制促進(jìn)肺癌的生長(zhǎng)和浸潤(rùn)轉(zhuǎn)移[23]。Brussino等[24]研究發(fā)現(xiàn)在NSCLC細(xì)胞中VEGF陽(yáng)性表達(dá)量較正常細(xì)胞明顯增高，本實(shí)驗(yàn)與Brussino等[24]的研究結(jié)果基本一致。此外，VEGF與NSCLC淋巴結(jié)的轉(zhuǎn)移密切相關(guān)，VEGF表達(dá)陽(yáng)性者，淋巴結(jié)轉(zhuǎn)移顯著增高[25]。同時(shí)還有研究表明，VEGF在許多腫瘤中的表達(dá)受一些miRNA調(diào)控，其中包括miR-200b[15，26-27]。但miR-200b與VEGF在肺癌中的表達(dá)調(diào)控未見報(bào)道。作為miR-200b的靶基因，VEGF是否參與了miR-200b對(duì)NSCLC細(xì)胞功能的調(diào)控？于是，本研究檢測(cè)了miR-200b與VEGF共同對(duì)NSCLC細(xì)胞增殖的影響。結(jié)果證實(shí)，miR-200b與VEGF共同高表達(dá)組細(xì)胞增殖率明顯高于miR-200b高表達(dá)組，miR-200b與VEGF共同表達(dá)抑制組細(xì)胞增殖率低于miR-200b高表達(dá)組。這說(shuō)明VEGF作為miR-200b的靶基因逆轉(zhuǎn)了miR-200b抑制NSCLC細(xì)胞增殖的作用。

本研究證實(shí)了NSCLC組織及細(xì)胞中miR-200b與VEGF表達(dá)呈負(fù)相關(guān)，并且VEGF可逆轉(zhuǎn)miR-200b對(duì)NSCLC細(xì)胞增殖抑制作用。但是VEGF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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VEGF reverses the inhibition of non-small cell lung cancer proliferation induced by miR-200b.

LUO Wei-min1, ZHANG Jun1,YU Zong-tao2,LIN Cheng-yi1,GUO Jia-long1.Department of Cardiothoracic Surgery1,Department of Clinical Laboratory2,the Affiliated Shiyan Taihe Hospital of Hubei University of Medicine,Shiyan 442000,Hubei,CHINA

ObjectiveTo investigate the function of vascular endothelial growth factor(VEGF)in the process of miR-200b inhibiting the proliferation of non-small cell lung cancer(NSCLC).MethodsSixty-five patients with NSCLC who underwent surgical treatment in our hospital from February 2013 to May 2014 were enrolled.Quantitative reverse transcription-PCR(qRT-PCR)was conducted to detect the expression of miR-200b and VEGF in NSCLC tissues.The correlation between miR-200b and VEGF in NSCLC tissues was statistically analyzed.A549,H460 and 16HBE cells were routinely cultured,the expression of miR-200b and VEGF in A549,H460 and 16HBE cells were detected by qRT-PCR.The normal cultured A549 cells were randomly divided into four groups:miR-200b mimics+vectorgroup,miR-200b mimics+VEGF group,miR-200b inhibitor+scramble group,miR-200b inhibitor+VEGF-siRNA group.miR-200b mimics+vector group and miR-200b inhibitor+scramble group were selected as the negative control. The tetrazolium-based colorimetric assay(MTT)was used to measure the OD value of each group.ResultsQRT-PCR showed that the expression levels of miR-200b and VEGF in NSCLC tissues were respectively(0.682±0.106)and (2.731±0.597),and the correlation analysis showed that miR-200b was negatively correlated with VEGF expression in NSCLC tissues(r=-0.514,P＜0.001).The expression of miR-200b in A549 and H460 cells was significantly lower than that in 16HBE cells(P=0.000),while the expression of VEGF in A549 and H460 cells was significantly higher than that in 16HBE cells(P=0.000).MTT showed that the proliferation rates of miR-200b mimics+VEGF group at 48 h and 72 h were significantly higher than that of miR-200b mimics+vector group(P＜0.05),while the cell proliferation rates of miR-200b inhibitor+VEGF-siRNA group were significantly lower than that of miR-200b inhibitor+scramble group at 48 h and 72 h(P＜0.05).ConclusionThe expression of miR-200b is negatively correlated with the expression of VEGF in NSCLC tissues and cells.VEGF reverses the effect of miR-200b in the proliferation of NSCLC cell.

miR-200b;Non-small cell lung cancer;Vascular endothelial growth factor(VEGF);Proliferation

R734.2

A

1003—6350（2017）01—0005—04

2016-07-19)

湖北省教育廳課題（編號(hào)：B2015477）

羅衛(wèi)民。E-mail：luoweiming0803@sina.com

10.3969/j.issn.1003-6350.2017.01.002