VEGF逆轉(zhuǎn)miR-200b促進非小細胞肺癌增殖的作用

羅衛(wèi)民，張軍，余宗濤，林稱意，郭家龍

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

VEGF逆轉(zhuǎn)miR-200b促進非小細胞肺癌增殖的作用

羅衛(wèi)民1，張軍1，余宗濤2，林稱意1，郭家龍1

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

目的探討血管內(nèi)皮生長因子(VEGF)在miR-200b抑制非小細胞肺癌(NSCLC)增殖中的作用及機制。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NSCLC組織中miR-200b與VEGF的表達，并統(tǒng)計分析NSCLC組織中miR-200b與VEGF表達的相關(guān)性；常規(guī)培養(yǎng)A549、H460及16HBE細胞，qRT-PCR檢測各組細胞中miR-200b與VEGF的表達；將常規(guī)培養(yǎng)的A549細胞隨機分為四組，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組作為陰性對照，采用MTT法檢測各組吸光度值(OD值)。結(jié)果qRT-PCR結(jié)果顯示，miR-200b在NSCLC組織的表達水平為(0.682± 0.106)，VEGF在NSCLC組織的表達水平為(2.731±0.597)，相關(guān)性分析顯示在NSCLC組織中miR-200b與VEGF的表達呈負相關(guān)(r=-0.514，P＜0.001)；miR-200b在A549、H460細胞中的表達顯著低于16HBE細胞，差異有統(tǒng)計學意義(P=0.000)，而VEGF在A549、H460細胞中的表達明顯高于16HBE細胞，差異有統(tǒng)計學意義（P=0.000）；MTT結(jié)果顯示，miR-200b mimics+VEGF組A549細胞在48 h、72 h的細胞增殖率明顯高于miR-200b mimics+vector組，miR-200b inhibitor+VEGF-siRNA組A549細胞在48 h、72 h的細胞增殖率明顯低于miR-200b inhibitor+scramble組，差異均有統(tǒng)計學意義(P＜0.05)。結(jié)論miR-200b與VEGF在NSCLC組織及細胞中表達呈負相關(guān)；VEGF逆轉(zhuǎn)miR-200b對NSCLC細胞增殖的作用。

miR-200b；非小細胞肺癌；血管內(nèi)皮生長因子；增殖

肺癌是全球死亡率最高的惡性腫瘤，70%～80%的肺癌是非小細胞肺癌(non samll cell lung cancer，NSCLC)，包括鱗狀細胞癌、腺癌、大細胞癌[1]。非小細胞肺癌的預后很差，盡管今年化療藥物研究進展及治療手段有所進步，NSCLC的5年總體生存率仍不超過11%。其主要死亡原因是化療耐藥性和轉(zhuǎn)移，但NSCLC轉(zhuǎn)移的機制仍不十分清楚[2]。

微小RNA(miRNAs)是一組包含19～25個核苷酸的非編碼小分子單鏈RNA，是基因表達的一個重要調(diào)控因子。通過與靶基因3′-UTR(3′-非編碼區(qū))形成完全或不完全地堿基配對，miRNA可導致靶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。miRNA同時也參與多種細胞生物學行為，包括細胞的分化、增殖、遷移、代謝及凋亡等過程。過去的研究已經(jīng)證明miRNA表達譜在不同的腫瘤中的作用，同時也表明了miRNA的表達異常與腫瘤的發(fā)生發(fā)展息息相關(guān)[3-4]。據(jù)報道，目前已有一些研究證實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中促進腫瘤的轉(zhuǎn)移，如miR-141[6]，miRNA-197[7]。miR-200b能抑制支氣管上皮細胞的上皮間葉轉(zhuǎn)化(EMT)及腫瘤細胞轉(zhuǎn)移[8]，同時miR-200b還能下調(diào)E盒結(jié)合鋅指蛋白2(ZEB2)及E2F3的表達，增加E-鈣連接素表達，從而抑制耐藥性NSCLC細胞增殖，并促進腫瘤細胞凋亡[9-10]。

血管內(nèi)皮生長因子(VEGF)是一種重要的血管生成促成因子，通過與特異性受體結(jié)合從而刺激血管內(nèi)皮細胞增殖，增加血管通透性，誘導腫瘤新生血管及淋巴管形成，其過度表達與腫瘤的形成、發(fā)展、轉(zhuǎn)移等病理過程密切相關(guān)[11]。研究表明VEGF在多種實體腫瘤中高表達，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通過靶向作用于VEGF及其受體負性調(diào)控VEGF信號通路，從而抑制腫瘤細胞血管的生成[15]。而NSCLC中，miR-200b與VEGF的表達調(diào)控關(guān)系尚無報道。本研究擬通過研究miR-200b在NSCLC中對VEGF表達調(diào)控的作用，探討miR-200b抑制NSCLC侵襲轉(zhuǎn)移的機制。

1 材料與方法

1.1 細胞培養(yǎng) NSCLC細胞株A549、H460及正常的肺支氣管上皮細胞系人支氣管上皮細胞(16HBE)，從美國標準生物品收藏中心(ATCC)購買。細胞培養(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 臨床標本收集及資料 人非小細胞肺癌取自本院2013年2月至2014年5月手術(shù)治療的65例患者，所有患者均無接受手術(shù)前化療。記錄患者年齡、性別、組織學分級、腫瘤大小、浸潤深度和淋巴結(jié)轉(zhuǎn)移等資料。本研究經(jīng)醫(yī)學倫理委員會批準。

1.3 試劑 miR-200b mimics、miR-200b inhibitor、VEGF過表達載體、vector、VEGF siRNA、scramble購自Genecopoeia公司。轉(zhuǎn)染試劑為Lipofectamine 2000，購自invitrogen公司。

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

1.5 細胞分組與轉(zhuǎn)染 將NSCLCA549細胞分為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組作為陰性對照。將A549細胞接種于6孔板，將上述轉(zhuǎn)染用的質(zhì)粒分別轉(zhuǎn)染細胞。根據(jù)轉(zhuǎn)染試劑說明書進行操作。

1.6 MTT實驗 將上述轉(zhuǎn)染48 h后的4組A549細胞接種于96孔板中，每組設(shè)5個復孔，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，選擇波長為570 nm，在酶標儀上測定各孔吸光值，記錄結(jié)果，實驗重復3次。

1.7 熒光實時定量PCR(quantitative real-time PCR，qRT-PCR)PCR擴增反應(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個循環(huán)。以β-actin為內(nèi)參，所測定的miR-200b的相對表達量采用2-ΔΔCT法分析。

1.8 統(tǒng)計學方法 采用SPSS13.0統(tǒng)計軟件對實驗數(shù)據(jù)進行分析，計量資料以均數(shù)±標準差(±s)表示，兩組間均數(shù)比較采用t檢驗，多組間均數(shù)比較采用方差分析，相關(guān)性分析用Pearson檢驗，以P＜0.05為差異有統(tǒng)計學意義。

2 結(jié) 果

2.1 miR-200b與VEGF在NSCLC組織及細胞中的表達 qRT-PCR檢測了NSCLC組織及細胞中miR-200b與VEGF的表達。結(jié)果顯示，miR-200b在NSCLC組織的表達水平為(0.682±0.106)，VEGF在NSCLC組織的表達水平為(2.731±0.597)，相關(guān)性分析顯示在NSCLC組織中miR-200b與VEGF的表達呈負相關(guān)(r=-0.514，P＜0.001)。miR-200b在NSCLC細胞中的表達顯著低于16HBE，差異有統(tǒng)計學意義(F= 214.670，P=0.000)，而NSCLC中VEGF的表達明顯高于16HBE，差異有統(tǒng)計學意義(F=107.820，P=0.000)，miR-200b及VEGF在NSCLC及16HBE細胞中的表達相反，見表1。

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

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

注：與16HBE細胞相比較，aP＜0.01。

16HBE細胞A549細胞2.158±0.573 0.740±0.092 0.536±0.084a2.905±0.746a

2.2 VEGF逆轉(zhuǎn)miR-200b對NSCLC細胞增殖的作用 MTT結(jié)果顯示，在轉(zhuǎn)染miR-200b mimics+ VEGF組的A549細胞48 h與72 h的OD值高于miR-200b mimics+vector組，差異有統(tǒng)計學意義；在轉(zhuǎn)染miR-200b inhibitor+VEGF-siRNA組的A549細胞48 h與72 h的OD值低于miR-200b inhibitor+scramble，差異有統(tǒng)計學意義(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NSCLC細胞增殖的影響(±s)

表2 miR-200b與VEGF對NSCLC細胞增殖的影響(±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，miRNAs在NSCLC發(fā)生發(fā)展過程中發(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)展的多個過程，包括細胞增殖、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癌基因抑制舌癌細胞上皮間質(zhì)轉(zhuǎn)化[18]，還能增強前列腺癌細胞對化療藥物的敏感性，抑制其增殖及轉(zhuǎn)移[19]。此外，miR-200b通過靶向Ras鳥苷酸結(jié)合蛋白超家族的新成員(RND3)來調(diào)節(jié)細胞周期蛋白D1(cyclin D1)表達水平從而促進宮頸癌細胞周期停滯[20]。在肺癌中，miR-200b通過靶向作用轉(zhuǎn)錄調(diào)控因子E2F3逆轉(zhuǎn)肺癌對多西他賽化療耐藥[9]，還能通過靶向血管內(nèi)皮生長因子受體1(FLT1/VEGFR1)抑制肺癌細胞遷移和增殖[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是生物活性最強的分子類型，而VEGF已被看作是許多不同惡性腫瘤生長和擴散轉(zhuǎn)移過程中的一個關(guān)鍵的致癌基因[22]。早期的研究發(fā)現(xiàn)，VEGF參與肺癌浸潤與轉(zhuǎn)移機制存在兩種形式，一種方式可能通過與內(nèi)皮細胞上的VEGFR結(jié)合，經(jīng)過旁分泌形式促進腫瘤新生血管形成及增加血管通透性從而導致肺癌細胞浸潤與轉(zhuǎn)移；另一種方式可能通過自分泌機制促進肺癌的生長和浸潤轉(zhuǎn)移[23]。Brussino等[24]研究發(fā)現(xiàn)在NSCLC細胞中VEGF陽性表達量較正常細胞明顯增高，本實驗與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表達陽性者，淋巴結(jié)轉(zhuǎn)移顯著增高[25]。同時還有研究表明，VEGF在許多腫瘤中的表達受一些miRNA調(diào)控，其中包括miR-200b[15，26-27]。但miR-200b與VEGF在肺癌中的表達調(diào)控未見報道。作為miR-200b的靶基因，VEGF是否參與了miR-200b對NSCLC細胞功能的調(diào)控？于是，本研究檢測了miR-200b與VEGF共同對NSCLC細胞增殖的影響。結(jié)果證實，miR-200b與VEGF共同高表達組細胞增殖率明顯高于miR-200b高表達組，miR-200b與VEGF共同表達抑制組細胞增殖率低于miR-200b高表達組。這說明VEGF作為miR-200b的靶基因逆轉(zhuǎn)了miR-200b抑制NSCLC細胞增殖的作用。

本研究證實了NSCLC組織及細胞中miR-200b與VEGF表達呈負相關(guān)，并且VEGF可逆轉(zhuǎn)miR-200b對NSCLC細胞增殖抑制作用。但是VEGF的這種作用調(diào)控的下游信號通路尚不清楚，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：B2015477）

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

10.3969/j.issn.1003-6350.2017.01.002