熊　瑋　駱　瑜　董少紅　李江華　廖碧紅　龐新利　羅林杰

(暨南大學(xué)第二臨床醫(yī)學(xué)院/深圳市人民醫(yī)院心內(nèi)科,深圳518020)

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微小RNA-146a上調(diào)cyclin D1表達(dá)誘導(dǎo)大鼠血管平滑肌細(xì)胞的增殖①

熊瑋駱瑜董少紅李江華廖碧紅龐新利羅林杰

(暨南大學(xué)第二臨床醫(yī)學(xué)院/深圳市人民醫(yī)院心內(nèi)科,深圳518020)

[摘要]目的：采用基因芯片技術(shù)研究小分子RNA-146a(miR-146a)促進(jìn)血管平滑肌細(xì)胞增殖的作用靶點(diǎn)并進(jìn)行驗(yàn)證。方法：原代培養(yǎng)大鼠血管平滑肌細(xì)胞，分成mimics組、inhibitor組、control組、sham組，分別體外轉(zhuǎn)染miR-146a mimics(50 nmol/L)、miR-146a inhibitor(50 nmol/L)、miR-146a錯(cuò)義鏈(50 nmol/L)、PBS，Real time PCR測(cè)定轉(zhuǎn)染后miR-146a水平，CCK8法檢測(cè)轉(zhuǎn)染后血管平滑肌細(xì)胞增殖情況。采用基因芯片檢測(cè)inhibitor組和control組基因表達(dá)譜，通過(guò)生物信息學(xué)技術(shù)篩選出差異基因和調(diào)控的信號(hào)通路。對(duì)篩選出來(lái)的信號(hào)通路用Real time PCR和Western blot進(jìn)行驗(yàn)證。結(jié)果：轉(zhuǎn)染48 h后，inhibitor組血管平滑肌細(xì)胞的miR-146a水平明顯低于control組和sham組(P<0.01)，inhibitor組血管平滑肌細(xì)胞的OD值明顯低于control組、sham組(P<0.05)。通過(guò)對(duì)基因表達(dá)譜分析發(fā)現(xiàn)，p53信號(hào)通路被miR-146a上調(diào)。用Real time PCR和Western blot進(jìn)行檢測(cè)發(fā)現(xiàn)，p53信號(hào)通路中關(guān)鍵分子p53、caspase3、PTEN的mRNA和蛋白水平無(wú)明顯變化(P>0.05)，而mimics組VSMC中cyclin D1的mRNA和蛋白水平增加(與sham相比，P<0.05)，inhibitor組VSMC中cyclin D1的mRNA和蛋白水平下降(與sham相比，P<0.05)。 結(jié)論：miR-146a可能通過(guò)上調(diào)細(xì)胞周期蛋白cyclin D1的表達(dá)促進(jìn)大鼠血管平滑肌細(xì)胞的增殖。

[關(guān)鍵詞]血管平滑肌細(xì)胞；miR-146a；基因譜；P53；cyclin D1

小分子RNA-146a(microRNA-146a,miR-146a)是第一個(gè)被發(fā)現(xiàn)在免疫系統(tǒng)中具有調(diào)節(jié)作用的小RNA，主要與類風(fēng)濕性關(guān)節(jié)炎、腫瘤、膿毒血癥等疾病的發(fā)生相關(guān)[1]。近年來(lái)的研究發(fā)現(xiàn)，急性冠脈綜合征患者外周血單個(gè)核細(xì)胞中miR-146a的水平顯著升高，miR-146a能促進(jìn)Th1細(xì)胞的分化，上調(diào)Th1細(xì)胞的功能，可能參與了對(duì)冠心病患者的免疫功能調(diào)節(jié)[2]。在頸動(dòng)脈球囊損傷后頸動(dòng)脈組織中miR-146a的表達(dá)水平顯著上調(diào)，miR-146a可能參與了球囊損傷后再狹窄的病理[3]。最近的研究還發(fā)現(xiàn)，miR-146a參與了ApoE-/-小鼠動(dòng)脈粥樣硬化的發(fā)生[4]。這些研究都表明miR-146a與動(dòng)脈粥樣硬化、冠心病和再狹窄的發(fā)生具有相關(guān)性，但它在這些疾病中的具體作用并不清楚。

在前期研究中，我們發(fā)現(xiàn)在血管平滑肌細(xì)胞(Vascular smooth muscle cell,VSMC)增殖過(guò)程中，miR-146a水平顯著上調(diào)，miR-146a可以促進(jìn)VSMC的增殖和遷移，抑制其凋亡，但具體機(jī)制不清楚[5-7]。本文擬通過(guò)基因芯片技術(shù)和信息學(xué)分析，探尋miR-146a促進(jìn)VSMC增殖的作用靶點(diǎn)和信號(hào)通路。

1材料與方法

1.1試劑與材料SPF級(jí)SD大鼠購(gòu)自廣東省實(shí)驗(yàn)動(dòng)物中心，DMEM培養(yǎng)基、0.25%胰酶、FBS購(gòu)自Gibco公司，Lipofectamine2000購(gòu)自Invitrogen公司，RNeasy Mini Kit試劑盒購(gòu)自Qiagen公司，All-in-one miRNA qRT-PCR detection kit購(gòu)自genecopoeia公司，SYBR Premix Ex Taq試劑盒購(gòu)自TaKaRa公司，CCK8試劑盒購(gòu)自同仁化學(xué)研究所，大鼠alpha-actin抗體購(gòu)自博士德公司，大鼠miR-146a mimics、抑制子及陰性對(duì)照由吉瑪公司合成，miR-146a、p53、caspase3、PTEN、cyclin D1、U6引物由生工公司合成，p53、caspase3、PTEN、cyclin D1抗體購(gòu)自Abcam公司。

1.2方法

1.2.1大鼠VSMC培養(yǎng)及miR-146a干擾按照本室建立的方法培養(yǎng)VSMC及進(jìn)行miR-146a干擾[5-7]。實(shí)驗(yàn)分為4組，高表達(dá)組(mimics)、抑制組(inhibitor)、陰性對(duì)照組(control)及正常VSMC組(sham)，其中miR-146a抑制子進(jìn)行6-FAM熒光標(biāo)記。分別轉(zhuǎn)染miR-146a mimics(50 nmol/L)、miR-146a抑制子(50 nmol/L)、miR-146a錯(cuò)義鏈(50 nmol/L)，sham組加入同等劑量PBS，5 h后換成完全培養(yǎng)基，熒光顯微鏡觀察轉(zhuǎn)染效率。

1.2.2熒光定量PCR轉(zhuǎn)染48 h后按照試劑盒指南操作，提取RNA逆轉(zhuǎn)錄成cDNA，并進(jìn)行PCR反應(yīng)。引物序列為：miR-146a上游引物(5′-3′)TGAGAACTGAATTCCATGGGTT，miR-146a下游引物為通用引物，U6上游引物(5′-3′)CTCGCTTCGGCAGCACA，U6下游引物(5′-3′)ACGCTTCACGAATTTGCGT，miR-146a與U6相對(duì)表達(dá)量用公式2-ΔΔCT計(jì)算。

1.2.3VSMC增殖細(xì)胞處理后以每孔104個(gè)細(xì)胞接種至96孔板，每組設(shè)6個(gè)復(fù)孔，24 h后進(jìn)行轉(zhuǎn)染，5 h后換為100 μl完全培養(yǎng)基，培養(yǎng)48 h后加入10 μl WST試劑染色，在37℃、5%CO2培養(yǎng)2 h，在酶聯(lián)免疫檢測(cè)儀下測(cè)定450 nm(參考波長(zhǎng)650 nm)的OD值。

1.2.4表達(dá)譜芯片檢測(cè)實(shí)驗(yàn)分成miR-146a抑制組及對(duì)照組，每組3個(gè)樣品，共6張芯片。具體檢測(cè)方法為轉(zhuǎn)染miR-146a inhibitor及control，48 h后吸出培養(yǎng)液，加入1～2 ml Trizol，-70℃保存送上?？党缮锕具M(jìn)行芯片檢測(cè)：樣品用Agilent ND1000檢測(cè)RNA是否降解及RNA濃度，用Agilent Quick Amp Labeling對(duì)樣品進(jìn)行標(biāo)記，Agilent Surehyb進(jìn)行雜交，用Agilent DNA Microarray Scanner進(jìn)行掃描，用Agilent Feature Extraction軟件(v11.0.1.1)采集芯片信號(hào)，用Agilent GeneSpring GX 12.1對(duì)原始數(shù)據(jù)進(jìn)行Quantile標(biāo)準(zhǔn)化和數(shù)據(jù)處理，兩組樣品間具有統(tǒng)計(jì)學(xué)意義的差異表達(dá)基因通過(guò)火山圖篩選和Fold Change篩選，使用標(biāo)準(zhǔn)的富集計(jì)算方法進(jìn)行GO分析和Pathway分析。

1.2.5實(shí)時(shí)PCR和Western 印跡分析將大鼠VSMC分成4組：sham、control、inhibitor及mimics，轉(zhuǎn)染48 h提取RNA，逆轉(zhuǎn)錄成cDNA，按照試劑盒方法進(jìn)行PCR反應(yīng)，檢測(cè)p53信號(hào)通路關(guān)鍵基因p53、caspase3、PTEN、cyclin D1的mRNA。

轉(zhuǎn)染48 h后提取總蛋白質(zhì)，每孔加入20 μl蛋白質(zhì)溶液，在10%的SDS-PAGE中80 V、30 min及120 V、1 h進(jìn)行電泳分離，半干轉(zhuǎn)液38 mA、90 min轉(zhuǎn)入PVDF膜，5%牛奶封閉2 h，加入兔抗大鼠p53一抗(1∶200)、caspase3一抗(1∶1 000)、PTEN一抗(1∶1 000)、cyclin D1一抗(1∶1 000)及GAPDH一抗(1∶1 000)4℃孵育過(guò)夜，1‰TBST洗10 min 3次，加入羊抗兔二抗(1∶6 000)孵育1 h，1‰TBST洗10 min 3次，加入ECL發(fā)光液100 μl在Image Quant RT ECL冷CCD成像系統(tǒng)進(jìn)行顯影，用Imagequant TL軟件進(jìn)行半定量分析。

2結(jié)果

2.1miR-146a促進(jìn)VSMC增殖轉(zhuǎn)染miR-146a 抑制子 5 h用熒光顯微鏡觀察，在VSMC胞漿內(nèi)可見(jiàn)大量顆粒狀熒光，表明miR-146a inhibitor已進(jìn)入VSMC內(nèi)(見(jiàn)圖1)。用實(shí)時(shí)PCR檢測(cè)3組細(xì)胞之間的miR-146a水平，與sham組和對(duì)照組相比，抑制子組的miR-146a mRNA相對(duì)表達(dá)水平顯著下降，差異具有顯著性(P<0.01)，sham組和control組相比miR-146a水平無(wú)明顯差異(P>0.05)，表明RNA干擾成功(見(jiàn)圖2A)。采用CCK8檢測(cè)干擾48 h時(shí)VSMC的增殖情況，sham組和control組OD值無(wú)明顯差異(P>0.05)，inhibitor組的OD值明顯下降(P<0.05)，表明miR-146a inhibitor抑制了VSMC的增殖(見(jiàn)圖2B)。

圖1　熒光顯微鏡檢測(cè)血管平滑肌細(xì)胞內(nèi)miR-146a抑制劑轉(zhuǎn)染效率Fig.1　miR-146a inhibitors labeled by 6-FAM fluorescence in VSMCs were detected by fluorescence microscope

圖2　miR-146a促進(jìn)血管平滑肌細(xì)胞增殖Fig.2　miR-146a promoted proliferation of VSMCsNote: A.Relative expression of miR-146a(Real time PCR);B.VSMCs proliferation(CCK8).Contrast with Sham,Δ.P<0.01,*.P<0.05.

2.2p53信號(hào)通路是miR-146a的作用靶點(diǎn)用實(shí)時(shí) PCR檢測(cè)inhibitor和control組之間的miR-146a相對(duì)水平，inhibitor組顯著低于control組(P<0.01)(見(jiàn)圖3)。在總共16 802個(gè)基因中，8 547個(gè)基因表達(dá)上調(diào)，占50.8%，8 255個(gè)基因表達(dá)下降，占49.2%.以變化倍數(shù)>2.0為截?cái)帱c(diǎn)，有806個(gè)基因上調(diào)，其中7個(gè)基因變化倍數(shù)>10.0(見(jiàn)表1)；在下調(diào)基因中有1 026個(gè)基因變化倍數(shù)>2.0，有9個(gè)基因變化倍數(shù)>10.0(見(jiàn)表2)。KEGG通路分析顯示P53信號(hào)通路被miR-146a上調(diào)。

2.3miR-146a上調(diào)cyclin D1表達(dá)Real time PCR結(jié)果顯示，各組VSMC之間p53、caspase3、PTEN的mRNA水平無(wú)明顯改變(P>0.05)，而mimics組VSMC中的cyclin D1 mRNA上調(diào)(sham vs mimics，P<0.001)，inhibitor組cyclin D1 mRNA水平下降(sham vs inhibitor，P<0.01)，sham與control組VSMC的cyclin D1相比無(wú)明顯差異(P>0.05)(見(jiàn)圖4A)。

Western印跡結(jié)果顯示，各組VSMC的p53、 caspase3、 PTEN蛋白的水平無(wú)明顯變化， 而 mimics

圖3　熒光定量PCR檢測(cè)miR-146a mRNA的相對(duì)水平Fig.3　Relative expression of miR-146a explored by real time PCRNote: Contrast with Control,*.P<0.01.

表1miR-146a上調(diào)且Fold change>10.0的基因

Tab.1Genes raised by miR-146a(Fold change>10.0)

GenesymbolGenbankaccessionFoldchangeP-valueFDRRegulationDdx60XM_00625306023.46518610.0006277180.007357081upNkx6-1NM_03173714.70960570.0191168130.051872179upBrinp3NM_17312113.07642470.0001254620.00347545upPlcd1NM_01703512.49469320.0004579920.006324951upPtpn5NM_01925312.36949960.0085046740.030104898upParp9NM_00110335111.18903980.0023357910.013887582upPrkg2NM_01301210.8583110.0014909770.010976278upDdx58NM_0011066459.17563010.0002059920.004289575up

表2miR-146a下調(diào)且Fold change>10.0的基因

Tab.2Genes decreased by miR-146a(Fold change>10.0)

GenesymbolGenbankaccessionFoldchangeP-valueFDRRegulationTbc1d31NM_00113484216.96458930.0013576080.010500376downCrabp1NM_00110571615.35712270.0006196420.007298685downSlfn3NM_05368713.31673960.0058545420.023927947downDchs1NM_00110754412.06812520.0002816760.004978058downLOC102555503XR_36076311.85774220.0314077960.073162969downSpdyaNM_13885511.52859130.0002543630.004785786downDlgap5NM_00113580210.63689990.0007633780.008067516downCse1lNM_00110860710.33489480.0008177590.008380534downTmem108XM_00622654310.2315780.0001090740.003233177down

圖4　miR-146a上調(diào)cyclin D1的表達(dá)水平Fig.4　miR-146a up-regulated the expression of cyclin D1Note: A.Relative expression of cyclin D1(Real time PCR)，contrast with Sham,#.P<0.001,*.P<0.01；B.Protein expression of p53,caspase3,PTEN,cyclin D1(Western blot).

組VSMC中的cyclin D1表達(dá)上調(diào)(sham vs mimics，P<0.05)，inhibitor組cyclin D1表達(dá)下降(sham vs inhibitor，P<0.05)，sham與control組VSMC的cyclin D1相比無(wú)明顯差異(P>0.05)(見(jiàn)圖4B)。

3討論

小分子RNA作為一個(gè)廣泛分布的重要的基因調(diào)節(jié)子，在機(jī)體的正常生長(zhǎng)、發(fā)育、分化、信號(hào)轉(zhuǎn)導(dǎo)、疾病和死亡等生理和病理過(guò)程均發(fā)揮重要作用。近年來(lái)的研究表明，miRNA不僅可以作為疾病的生物學(xué)診斷標(biāo)志用于疾病的診斷，還可以作為基因藥物用于疾病的治療[8,9]。

miR-146a被認(rèn)為是調(diào)節(jié)免疫功能的主要miRNA之一[10]。研究發(fā)現(xiàn)，用脂多糖刺激單核細(xì)胞，可以誘導(dǎo)miR-146a表達(dá)增加，用腫瘤壞死因子-α(Tumor necrosis factor,TNF-α)和白細(xì)胞介素-1β(Interleukin-1,IL-1β)也能夠通過(guò)核因子(Nuclear factor-κB,NF-κB)依賴的途徑促進(jìn)miR-146a的表達(dá)。miR-146a通過(guò)下調(diào)TNF受體相關(guān)因子6和白介素1受體相關(guān)激酶1的水平，負(fù)調(diào)控炎癥和免疫反應(yīng)，避免過(guò)度炎癥反應(yīng)的發(fā)生[11，12]。在類風(fēng)濕性關(guān)節(jié)炎、腫瘤、膿毒血癥等疾病的研究中也證實(shí)了miR-146a在疾病發(fā)生發(fā)展中的重要作用，可以作為某些疾病的生物學(xué)標(biāo)志[13-15]。Guo等[2]發(fā)現(xiàn)急性冠脈綜合征患者外周血單個(gè)核細(xì)胞中miR-146a的水平顯著升高，并能上調(diào)Th1細(xì)胞的功能，給予miR-146a可以促進(jìn)Th1細(xì)胞的分化和NF-κBp65的合成，表明miRNA-146a可能與冠心病患者的免疫功能調(diào)節(jié)有關(guān)。也有學(xué)者發(fā)現(xiàn)，在球囊損傷再狹窄的動(dòng)物模型中miR-146a的表達(dá)水平顯著上調(diào)[3]，可能參與了動(dòng)脈粥樣硬化和球囊損傷后再狹窄的發(fā)生發(fā)展過(guò)程。載脂蛋白E(ApoE)是調(diào)節(jié)脂質(zhì)代謝的重要蛋白，對(duì)動(dòng)脈粥樣硬化發(fā)揮負(fù)調(diào)控作用。最近的研究發(fā)現(xiàn)，在單核細(xì)胞和巨噬細(xì)胞中ApoE通過(guò)促進(jìn)miR-146a表達(dá)，抑制NF-κB，抑制炎癥和動(dòng)脈粥樣硬化的發(fā)生[4]。在急性ST抬高型心肌梗死的患者中循環(huán)miR-146a有助于預(yù)測(cè)心室重構(gòu)的發(fā)生[16]。這些研究表明miR-146a與動(dòng)脈粥樣硬化、冠心病、再狹窄等疾病存在密切關(guān)聯(lián)，但研究結(jié)果存在不一致。如Guo等[2]發(fā)現(xiàn)miR-146a促進(jìn)Th1細(xì)胞合成NF-κB，而在巨噬細(xì)胞和單核細(xì)胞中miR-146a抑制NF-κB表達(dá)[4]。

我們?cè)谇捌谘芯恐邪l(fā)現(xiàn)，在血管平滑肌細(xì)胞增殖過(guò)程中miR-146a水平顯著上調(diào)；用RNA干擾的方法抑制VSMC中miR-146a表達(dá)后，VSMC增殖和遷移明顯減少，凋亡增加，但具體機(jī)制不清楚[5-7]。雙熒光素酶報(bào)告基因檢測(cè)是研究miRNA的目的基因的經(jīng)典方法，但該方法對(duì)信號(hào)通路的研究缺乏系統(tǒng)性。在本文中，我們采用了基因表達(dá)譜芯片來(lái)篩選miR-146a發(fā)生作用所依賴的靶點(diǎn)和信號(hào)通路，檢測(cè)結(jié)果和生物信息學(xué)分析顯示p53信號(hào)通路被miR-146a inhibitor激活。進(jìn)一步研究發(fā)現(xiàn)，p53信號(hào)通路中的關(guān)鍵分子cyclin D1的基因和蛋白水平均被miR-146a上調(diào)。

細(xì)胞周期蛋白(cyclin)和細(xì)胞周期蛋白依賴性激酶(CKD)的復(fù)合物(cyclin/CDKs)在細(xì)胞從G1期向S期轉(zhuǎn)化的過(guò)程中發(fā)揮重要調(diào)控作用，其中最關(guān)鍵的調(diào)控蛋白為cyclin D1/CDK4[17]。多個(gè)研究都發(fā)現(xiàn)cyclin D1活化與血管平滑肌細(xì)胞的增殖和血管內(nèi)膜的重構(gòu)密切相關(guān)，是介導(dǎo)血管平滑肌細(xì)胞增殖的重要作用靶點(diǎn)[18-20]。在本文中，我們發(fā)現(xiàn)miR-146a可能是通過(guò)上調(diào)cyclin D1的表達(dá)促進(jìn)了血管平滑肌細(xì)胞的增殖。

參考文獻(xiàn)：

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[收稿2015-10-15修回2016-01-13]

(編輯許四平)

doi:10.3969/j.issn.1000-484X.2016.07.009

作者簡(jiǎn)介：熊瑋(1975年-)，男，博士，副主任醫(yī)師，主要從事冠心病的基礎(chǔ)研究，E-mail:xw0926@126.com。

中圖分類號(hào)R544.1

文獻(xiàn)標(biāo)志碼A

文章編號(hào)1000-484X(2016)07-0974-05

MicroRNA-146a promotes proliferation of rat vascular smooth muscle cells by up-regulating cyclin D1 expression

XIONG Wei,LUO Yu,DONG Shao-Hong,LI Jiang-Hua,LIAO Bi-Hong,PANG Xin-Li,LUO Lin-Jie.

Department of Cardiology，the Second Clinical Medical College of Ji′nan University,Shenzhen People′s Hospital,Shenzhen 518020,China

[Abstract]Objective:To detect and verifica the gene profile difference of microRNA-146a (miR-146a) and its role in the proliferation of vascular smooth muscle cells (VSMCs) by gene chip technology.Methods: Artificially synthesized miR-146a mimics(50 nmol/L),miR-146 inhibitor(50 nmol/L),scramble(50 nmol/L) and PBS were transfected into cultured primary rat VSMCs in vitro.After transfection,Real time PCR was used to measure the levels of miR-146a and the cell counting kit 8(CCK8) was employed to investigate the proliferation of VSMCs.The VSMCs interfered by miR-146a inhibitor or miR-146a control were examined by gene chips and the profile of gene were analyzed by bioinformatics technology to detect the different genes and signal transduction pathway.The changes in mRNAs and proteins were accessed separately by Real time PCR and Western blot.Results: Compared with sham and control VSMCs,miR-146a expression level was significantly decreased in treatment with miR-146a inhibitor(P<0.01),as well as optical density(OD) was also shown remarkably down regulated simultaneously(P<0.05).The investigation of gene profile revealed that the p53 signal pathway was up-regulated in VSMCs interfered by miR-146a.The mRNA and protein expression levels of p53,caspase3 and PTEN in p53 signal transduction pathway didn′t show significant differences(P>0.05),however,the mRNA and protein expression levels of cyclin D1 significantly increased in treatment with miR-146a mimics VSMCs group and decreased in miR-146a inhibitor VSMCs group(compared with sham VSMCs group,both P<0.05).Conclusion: Our data indicated that miR-146a may promote the proliferation of rat VSMCs by up-regulating cyclin D1 expression.

[Key words]Vascular smooth muscle cell;miRNA-146a;Gene profile; p53;cyclin D1

①本文為深圳市衛(wèi)計(jì)委資助課題(No.201505001)。