萬春花， 蔡 鈞， 楊世疆， 吳景冬， 郭清皓， 蔣林濤△

華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬武漢中心醫(yī)院 1麻醉科 2急診創(chuàng)傷外科，武漢 430014

miR-186對(duì)膠質(zhì)母細(xì)胞瘤細(xì)胞增殖和凋亡的影響及機(jī)制

萬春花1， 蔡 鈞2， 楊世疆2， 吳景冬2， 郭清皓2， 蔣林濤2△

華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬武漢中心醫(yī)院1麻醉科2急診創(chuàng)傷外科，武漢 430014

目的研究轉(zhuǎn)染miR-186對(duì)膠質(zhì)母細(xì)胞瘤細(xì)胞增殖能力的影響并探討作用機(jī)制。方法采用熒光定量PCR檢測(cè)miR-186在膠質(zhì)母細(xì)胞瘤細(xì)胞系U251、U87-MG及正常星形膠質(zhì)細(xì)胞系HA1800中的表達(dá)水平，將U251細(xì)胞分為miR-186模擬物組和對(duì)照組，分別轉(zhuǎn)染miR-186模擬物和陰性隨機(jī)對(duì)照序列，并用熒光定量PCR檢測(cè)miR-186在兩組中的表達(dá)量。采用CKK-8法檢測(cè)兩組細(xì)胞細(xì)胞增殖能力，流式細(xì)胞儀測(cè)定兩組細(xì)胞凋亡率，Western blot分析Caspase-3、Caspase-8、Caspase-9及Cyclin D1蛋白的表達(dá)。結(jié)果miR-186低表達(dá)于膠質(zhì)母細(xì)胞瘤細(xì)胞系U251及U87-MG，高表達(dá)于正常星形膠質(zhì)細(xì)胞系HA1800。轉(zhuǎn)染24、48、72及96 h后，miR-186模擬物組細(xì)胞增殖(A450nm值)低于對(duì)照組(均P<0.05)。轉(zhuǎn)染48 h后，miR-186模擬物組凋亡率高于對(duì)照組；miR-186模擬物組Caspase-3、9的表達(dá)量高于對(duì)照組(均P<0.01)，Caspase-8與對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。miR-186模擬物組Cyclin D1表達(dá)水平較對(duì)照組明顯降低(P<0.01)。結(jié)論miR-186抑制膠質(zhì)母細(xì)胞瘤細(xì)胞增殖能力，并誘導(dǎo)凋亡，其機(jī)制可能與上調(diào)Caspase-3、9，下調(diào)Cyclin D1有關(guān)。

miR-186； 膠質(zhì)母細(xì)胞瘤； 增殖； 細(xì)胞凋亡

膠質(zhì)母細(xì)胞瘤是成年人最常見的原發(fā)性腦部腫瘤，具有很強(qiáng)的侵襲和增殖能力，病死率很高，患者中位生存期一般不超過1年[1-2]。MicroRNAs (miRNA)是一類內(nèi)源性的非編碼RNA，長(zhǎng)度在21～25 nt，具有在轉(zhuǎn)錄后水平調(diào)控靶基因表達(dá)的功能[3-4]。miRNA參與調(diào)節(jié)細(xì)胞生長(zhǎng)、增殖、凋亡，與腫瘤的發(fā)生和發(fā)展有著密切關(guān)系[5]。miRNA-186被證實(shí)在神經(jīng)內(nèi)分泌腫瘤患者腫瘤組織中表達(dá)水平降低，且參與神經(jīng)內(nèi)分泌腫瘤增殖、浸潤(rùn)、轉(zhuǎn)移等過程[6]。它通過靶向SKP2抑制食管鱗狀細(xì)胞癌細(xì)胞增殖，并誘導(dǎo)凋亡[7]。miRNA-186在非小細(xì)胞肺癌中通過靶向結(jié)合MAP3K2基因而發(fā)揮抑制腫瘤細(xì)胞增殖和侵襲的作用[8]。細(xì)胞周期失調(diào)是發(fā)生癌變的重要原因，細(xì)胞周期蛋白D1(Cyclin D1)是G1期所必須的核蛋白，是G1/S期關(guān)鍵限速點(diǎn)的重要調(diào)節(jié)因子[9-10]。細(xì)胞凋亡是細(xì)胞死亡的調(diào)節(jié)性生理過程，Caspase引發(fā)的一系列級(jí)聯(lián)反應(yīng)被認(rèn)為是細(xì)胞凋亡過程的重要途徑[11]。本研究觀察了miR-186基因轉(zhuǎn)染對(duì)膠質(zhì)母細(xì)胞瘤U251細(xì)胞系增殖和凋亡的影響，分析了miR-186對(duì)細(xì)胞周期蛋白Cyclin D1及Caspase家族重要成員3、8、9的調(diào)控作用，探討miR-186在膠質(zhì)母細(xì)胞瘤中的作用機(jī)制。

1 材料與方法

1.1 主要實(shí)驗(yàn)材料

膠質(zhì)母細(xì)胞瘤細(xì)胞系U251、U87-MG及正常星形膠質(zhì)細(xì)胞系HA1800購(gòu)自中國(guó)醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)醫(yī)學(xué)中心，DMEM細(xì)胞培養(yǎng)液、胎牛血清、胰蛋白酶均購(gòu)自美國(guó)Hyclone公司，實(shí)驗(yàn)所需的一抗購(gòu)自美國(guó)Cell Signaling公司，二抗購(gòu)自武漢博士德生物科技有限公司， miRcute miRNA提取分離試劑盒(DP501)購(gòu)自北京天根生物科技有限公司，All-in-OneTMmiRNA qRT-PCR 檢測(cè)試劑盒購(gòu)自北京亞太恒信生物科技有限公司，SYBR Green Reagents 購(gòu)自TaKaRa公司，Caspase-3、8、9活性檢測(cè)試劑盒購(gòu)自碧云天生物科技有限公司，miR-186 mimics及scramble均由上海吉瑪生物科技有限公司合成。

1.2 細(xì)胞培養(yǎng)、轉(zhuǎn)染及分組

膠質(zhì)母細(xì)胞瘤細(xì)胞系U251、U87-MG及正常星形膠質(zhì)細(xì)胞系HA1800均加入DMEM培養(yǎng)液，于37℃、5%CO2培養(yǎng)箱中培養(yǎng)，培養(yǎng)48 h后消化傳代。將膠質(zhì)母細(xì)胞瘤細(xì)胞系U251分為miR-186模擬物組和對(duì)照組，分別轉(zhuǎn)染miR-186 mimics和陰性隨機(jī)對(duì)照序列scramble,采用Lipofectamine 2000 reagent (Invitrogen,USA)對(duì)兩組進(jìn)行轉(zhuǎn)染。miR-186模擬物組轉(zhuǎn)染序列：miR-186 mimics上游 5′-CAAAGAAUUCUCCUUUUGGGCU-3′,miR-186 mimics下游 5′-CCCAAAAGGAGAAUUCUUUG-UU-3′；對(duì)照組轉(zhuǎn)染序列：miR-186 NC上游 5′-UUCUCCGAACGUGUCACGUTT-3′,miR-186 NC下游 5′-ACGUGACACGUUCGGAGAATT-3′。

1.3 miRNA提取及qRT-PCR檢測(cè)

用miRcute miRNA提取分離試劑盒按操作說明書提取細(xì)胞系中miRNA ，采用All-in-OneTMmiRNA qRT-PCR 檢測(cè)試劑盒進(jìn)行反應(yīng)，操作按試劑盒說明書進(jìn)行。反應(yīng)體系：2×All-in-OneTMqRT-PCR Mix 10 μL、引物序列(10 μmol/L)1 μL、反轉(zhuǎn)錄cDNA 1 μL，加雙氧水至20 μL。反應(yīng)條件：預(yù)變性95℃ 10 min、變性95℃ 10 s、退火60℃ 20 s、延伸72℃ 30 s，共35周期。miR-181引物序列：上游5′-TGCGGGTGCTCCGCTTCGGCAGC-3′，下游5′-CAGTGCAGGGTCCGAGGT-3′，U6小核引物序列：上游5′-CGCAAGGATGACACG-3′；下游5′-GAGCAGGCTGGAGAA-3′。采用ABI Prism 7700型熒光實(shí)時(shí)定量PCR儀進(jìn)行檢測(cè)分析，以U6小核RNA作為內(nèi)參，2-ΔΔCt法定量分析miR-186相對(duì)表達(dá)水平。

1.4 細(xì)胞增殖能力測(cè)定

采用CCK-8法測(cè)定兩組細(xì)胞增殖能力。將miR-186模擬物組和對(duì)照組兩組細(xì)胞消化成單細(xì)胞懸液后，在96孔板上以2×103個(gè)/孔接種，每孔培養(yǎng)液體積200 μL。在分別培養(yǎng)0、24、48、72 、96 h后，每孔加入20 μL CCK-8溶液，繼續(xù)培養(yǎng)1 h后，用酶標(biāo)儀在450 nm波長(zhǎng)下測(cè)定各孔吸光值(A)，以時(shí)間為橫坐標(biāo)，吸光值為縱坐標(biāo)繪制膠質(zhì)瘤細(xì)胞增殖曲線。

1.5 細(xì)胞凋亡能力測(cè)定

采用流式細(xì)胞術(shù)測(cè)定兩組細(xì)胞凋亡能力。用Annexin Ⅴ/PI 染色檢測(cè)，將兩組細(xì)胞消化成單細(xì)胞懸液后，PBS清洗2次，并使用Binding Buffer重懸，加入相應(yīng)比例的Annexin Ⅴ抗體，避光染色10 min后加入適量PBS溶液以及PI染料，流式細(xì)胞儀檢測(cè)Annexin Ⅴ陽(yáng)性細(xì)胞比例來確定細(xì)胞凋亡的變化。

1.6 Caspase-3、8、9酶活性測(cè)定

將miR-186模擬物組和對(duì)照組兩組細(xì)胞裂解后，離心處理(1 000 r/min,5min)，按Caspase-3、8、9活性檢測(cè)試劑盒說明書進(jìn)行檢測(cè)。利用96孔板，將細(xì)胞裂解液與分別含有Caspase-3、8、9底物的反應(yīng)液共同孵育4 h，然后用TECAN酶標(biāo)儀讀取405 nm的吸光度值，計(jì)算相對(duì)活力值。

1.7 Cyclin D1蛋白表達(dá)量測(cè)定

采用Western blot法。將miR-186模擬物組和對(duì)照組兩組細(xì)胞用RIPA細(xì)胞裂解液冰上裂解30 min后，變性、上樣，以每孔30 μg總蛋白上樣，濃縮膠80 V電泳40 min，分離膠100 V電泳2 h。常規(guī)濕法轉(zhuǎn)膜，加入Cyclin D1一抗(1∶300)，37℃孵育4 h，二抗(1∶500)孵育過夜， ECL液顯影，Quantity One 1-D分析軟件對(duì)蛋白質(zhì)印跡條帶進(jìn)行定量。目的蛋白相對(duì)表達(dá)量=目的蛋白測(cè)定值/ GAPDH測(cè)定值，實(shí)驗(yàn)重復(fù)3次，取平均值。

1.8 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 miR-186低表達(dá)于膠質(zhì)母細(xì)胞瘤細(xì)胞系

熒光定量PCR結(jié)果示，以正常星形膠質(zhì)細(xì)胞系HA1800中 miR-186相對(duì)表達(dá)量為1.0，miR-186在膠質(zhì)母細(xì)胞瘤細(xì)胞系U251、U87-MG中的相對(duì)表達(dá)量分別是(0.20±0.04)、(0.30±0.05)，明顯低于正常星形膠質(zhì)細(xì)胞系HA1800(均P<0.01)，見圖1。

與HA1800細(xì)胞比較，**P<0.01圖1 miR-186在膠質(zhì)母細(xì)胞瘤細(xì)胞系U251、U87-MG及正常星形膠質(zhì)細(xì)胞系中的表達(dá)Fig.1 The expression of miR-186 in glioblastoma cell line,U251 and U87-MG,and normal astrocyte cell line HA1800

2.2 miR-186過表達(dá)抑制膠質(zhì)母細(xì)胞瘤細(xì)胞增殖

轉(zhuǎn)染miR-186 mimics 24 h后，熒光定量PCR結(jié)果示，以陰性對(duì)照組中miR-186表達(dá)量為1.0，膠質(zhì)母細(xì)胞瘤系U251中miR-186的相對(duì)表達(dá)量是(8.00±0.63)，明顯高于陰性對(duì)照組(P<0.01)，見圖2A。

采用CCK-8法測(cè)定U251細(xì)胞增殖。轉(zhuǎn)染后0、24、48、72及96 h后，miR-186模擬物組與對(duì)照組的A450nm值分別為(0.33±0.03)vs.(0.35±0.04)、(0.43±0.08)vs.(0.75±0.10)、(0.72±0.09)vs.(1.67±0.25)、(1.33±0.15)vs.(2.43±0.19)及(1.92±0.20)vs.(3.78±0.32) ，除0 h時(shí)兩組差異無統(tǒng)計(jì)學(xué)意義外，其余時(shí)間點(diǎn)差異均有統(tǒng)計(jì)學(xué)意義(P<0.05，P<0.01)，見圖2B。

2.3 miR-186過表達(dá)誘導(dǎo)膠質(zhì)母細(xì)胞瘤細(xì)胞凋亡

轉(zhuǎn)染miR-186模擬物48 h后，流式細(xì)胞術(shù)測(cè)定兩組細(xì)胞的凋亡率。miR-186模擬物組凋亡率為 (20.0±1.5)%，陰性對(duì)照組凋亡率為 (8.0±1.0) %， miR-186模擬物組凋亡率高于對(duì)照組(P<0.01)，見圖3A。

A：miR-186在miR-186模擬物組及陰性對(duì)照組中的表達(dá)；B：CCK-8實(shí)驗(yàn)示miR-186模擬物組及陰性對(duì)照組的增殖曲線；與陰性對(duì)照組比較，*P<0.05 **P<0.01圖2 miR-186過表達(dá)抑制膠質(zhì)母細(xì)胞瘤細(xì)胞增殖Fig.2 Over-expression of miR-186 inhibits the proliferation of glioblastoma cells

Caspase-3、8、9活性檢測(cè)試劑盒檢測(cè)Caspase-3、8、9活力。分別以陰性對(duì)照組Caspase-3、8、9的活力值為1.0，miR-186模擬物組Caspase-3、9的相對(duì)活力分別為(2.1±0.2)、(2.2±0.1)，均顯著高于陰性對(duì)照組(均P<0.01)，而Caspase-8相對(duì)活力為(1.1±0.1)，與對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義(P>0.05)，見圖3B。

A：miR-186模擬物組與陰性對(duì)照組凋亡率的比較；B：miR-186模擬物組與陰性對(duì)照組Caspase-3、8、9活性比較；與陰性對(duì)照組比較，**P<0.01圖3 miR-186過表達(dá)誘導(dǎo)膠質(zhì)母細(xì)胞瘤細(xì)胞凋亡Fig.3 Over-expression of miR-186 induces the apoptosis of glioblastoma cells

2.4 miR-186下調(diào)Cyclin D1表達(dá)

Western blot結(jié)果示，以陰性對(duì)照組為1.0，miR-186模擬物組Cyclin D1相對(duì)表達(dá)量為(0.33±0.05)，明顯低于陰性對(duì)照組(P<0.01)，見圖4。

A：Western blot示miR-186模擬物組與陰性對(duì)照組Cyclin D1的表達(dá)；B：miR-186模擬物組與陰性對(duì)照組Cyclin D1相對(duì)表達(dá)量的比較；與陰性對(duì)照組比較，**P<0.01圖4 miR-186下調(diào)Cyclin D1表達(dá)Fig.4 miR-186 down-regulates expression of Cyclin D1

3 討論

膠質(zhì)母細(xì)胞瘤是最常見的原發(fā)性惡性腫瘤，年發(fā)病率約為5.26/10萬，每年新增患者約17 000人[12-13]。其發(fā)病機(jī)制尚不清楚，目前治療上仍以手術(shù)切除、放療同時(shí)聯(lián)合替莫唑胺化療為主，但患者普遍預(yù)后差、生活質(zhì)量低、生存期短[14]。miRNA是一種內(nèi)源性非編碼小RNA，在調(diào)控細(xì)胞增殖、分化、凋亡及多種信號(hào)途徑中起重要作用，參與腫瘤的發(fā)生和發(fā)展[15]。比如，miR-21通過調(diào)控程序性細(xì)胞死亡因子參與乳腺癌細(xì)胞增殖[16]；miR-34a通過直接抑制CD44抑制前列腺癌干細(xì)胞的轉(zhuǎn)移[17]。miR-186也被證實(shí)與腫瘤的發(fā)生有密切聯(lián)系，miR-186干擾肺腺癌細(xì)胞周期進(jìn)程，且與預(yù)后不良有關(guān)[18]；miR-186調(diào)控PTTG1表達(dá)促進(jìn)非小細(xì)胞肺癌遷移和侵襲[19]；miR-186靶向NSBP1抑制膀胱癌的生長(zhǎng)和轉(zhuǎn)移[20]。但是，miR-186在膠質(zhì)母細(xì)胞瘤中的作用尚不清楚。

細(xì)胞凋亡是導(dǎo)致細(xì)胞程序性死亡的特定過程，通過激活線粒體通路或死亡受體通路或內(nèi)質(zhì)網(wǎng)通路等途徑而誘發(fā)[21-22]。線粒體介導(dǎo)的細(xì)胞凋亡通路通過激活細(xì)胞色素C釋放到細(xì)胞質(zhì)中，與Apaf1結(jié)合形成復(fù)合物，引起Caspase-9等級(jí)聯(lián)反應(yīng)，最后導(dǎo)致Caspase-3的激活，繼而導(dǎo)致凋亡的發(fā)生[23]。本研究發(fā)現(xiàn)，miR-186模擬物組細(xì)胞凋亡率是(20.0±1.5)%,而對(duì)照組是(8.0±1.0)%，表明miR-186上調(diào)表達(dá)可誘導(dǎo)膠質(zhì)母細(xì)胞瘤細(xì)胞凋亡。通過檢測(cè)蛋白活力發(fā)現(xiàn)，miR-186模擬物組中Caspase-3和Caspase-9蛋白活力顯著升高。故而證實(shí)miR-186過表達(dá)激活了線粒體凋亡途徑進(jìn)而促進(jìn)膠質(zhì)母細(xì)胞瘤U251細(xì)胞凋亡。

死亡受體如Fas和腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(TRAIL)，通過招募Fas相關(guān)死亡結(jié)構(gòu)域和Caspase-8同型二聚體而自動(dòng)酶解，Caspase-8被激活并釋放到胞質(zhì)中，作用于Caspase-3并使Caspase-3激活，從而誘導(dǎo)凋亡[24]。本研究中，蛋白活力檢測(cè)表明miR-186模擬物組與對(duì)照組中Caspase-8蛋白活力無顯著差異，表明細(xì)胞表面死亡受體途徑?jīng)]有參與到miR-186誘導(dǎo)的膠質(zhì)母細(xì)胞瘤U251細(xì)胞凋亡過程。

細(xì)胞周期紊亂是導(dǎo)致惡性腫瘤轉(zhuǎn)化和失控性增殖的重要原因[25]。細(xì)胞周期調(diào)控激酶復(fù)合物CDK/Cyclin是調(diào)控細(xì)胞周期的關(guān)鍵因素，G0/G1期阻滯是抑制細(xì)胞增殖的重要原因[26-28]。本研究也探討了miR-186過表達(dá)是否引起U251細(xì)胞阻滯于G0/G1期。Western blot結(jié)果顯示，miR-186模擬物組中Cyclin D1(G1/S轉(zhuǎn)換期的重要正向調(diào)控因子[29])的表達(dá)水平較對(duì)照組明顯下降。這表明，miR-186基因過量表達(dá)引起U251細(xì)胞阻滯于G0/G1期，抑制細(xì)胞的有絲分裂，進(jìn)而抑制膠質(zhì)母細(xì)胞瘤增殖過程。

綜上，本研究發(fā)現(xiàn)轉(zhuǎn)染miR-186后，能顯著降低膠質(zhì)母細(xì)胞瘤U251細(xì)胞的增殖能力，并促進(jìn)凋亡，顯著提高細(xì)胞凋亡蛋白酶Caspase-3、Caspase-9的活力，降低細(xì)胞周期蛋白Cyclin D1的表達(dá)。說明，miR-186通過參與調(diào)控細(xì)胞凋亡和細(xì)胞周期途徑而抑制膠質(zhì)母細(xì)胞瘤U251的增殖能力。miR-186有望成為膠質(zhì)母細(xì)胞瘤治療的潛在標(biāo)志物，為腫瘤治療提供了新靶點(diǎn)及思路。

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EffectofmiR-186onProliferationandApoptosisofGliblastomaCellsandItsMechanisms

Wan Chunhua1, Cai Jun2, Yang Shijiang2etal

1DepartmentofAnesthesiology2DepartmentofEmergencyTraumaSurgery,WuhanCentralHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan430014,China

ObjectiveTo explore the role of miR-186 transfection in the proliferation and apoptosis of glioblastoma cells,as well as the regulatory mechanism.MethodsReal-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression of miR-186 in glioblastoma cell line,U251 and U87-MG,and normal astrocyte cell line HA1800.The U251 cells were divided into miR-186 mimic group and control group,which were transfected with miR-186 mimics and scramble sequence,respectively.MiR-186 expression levels of these two groups were detected by qRT-PCR.Cell proliferation ability of the two groups was evaluated by CKK-8 assay.Cell apoptosis rate was analyzed by flow cytometry.Caspase-3,Caspase-8,Caspase-9,and Cyclin D1 protein levels were quantified by Western blotting.ResultsLower miR-186 levels were observed in glioblastoma U251 and U87-MG cells than in normal brain cell line.However,miR-186 level was significantly higher in miR-186 mimic group than in control group (P<0.01).After transfection for 24,48,72,and 96 h,A450 nmvalues were lower in the miR-186 mimic group than in the control group (allP<0.05).After transfection for 48 h,apoptosis rate was higher in the miR-186 mimic group than in the control group.Caspase-3 and Caspase-9 were significantly higher in the miR-186 mimic group than in the control group (bothP<0.01).However,there was no significant difference in the expression of Caspase-8 between miR-186 mimic group and control group(P>0.05).Cyclin D1 protein expression was significantly lower in the miR-186 mimic group than in the control group (P<0.01).ConclusionMiR-186 inhibits the proliferation and induces apoptosis of U251 cells via up-regulating Caspase-3 and 9 and down-regulating Cyclin D1.

miR-186; glioblastoma; proliferation; apoptosis

萬春花，女，1977年生，主治醫(yī)師，E-mail:wanchunhuahp@tom.com

△通訊作者，Correspondina author，E-mail:lintaojiang99@21cn.com

R739.41

10.3870/j.issn.1672-0741.2017.06.005

(2017-05-17 收稿)