朱融和,孫媛媛,錢燕

(溫州醫(yī)科大學(xué)附屬第一醫(yī)院 兒科,浙江 溫州 325015)

MiR-152對轉(zhuǎn)化生長因子β1誘導(dǎo)的氣管上皮細(xì)胞向間質(zhì)細(xì)胞轉(zhuǎn)化的影響

朱融和,孫媛媛,錢燕

(溫州醫(yī)科大學(xué)附屬第一醫(yī)院 兒科,浙江 溫州 325015)

目的:研究miR-152在轉(zhuǎn)化生長因子β1(TGF-β1)誘導(dǎo)的氣管上皮細(xì)胞向間質(zhì)細(xì)胞轉(zhuǎn)化(EMT)中的表達(dá)情況,探討miR-152能否影響EMT的發(fā)生.方法:用10 ng/mL的TGF-β1誘導(dǎo)人氣管上皮16HBE細(xì)胞EMT發(fā)生,光鏡下觀察細(xì)胞形態(tài)變化,qRT-PCR和Western blot檢測細(xì)胞中EMT相關(guān)標(biāo)志物α平滑肌肌動蛋白(α-SMA)、E-cadherin和Vimentin的改變;qRT-PCR檢測有或無TGF-β1處理的16HBE細(xì)胞中miR-152的表達(dá)情況;通過轉(zhuǎn)染miR-152 mimics和miR-NC至細(xì)胞后,再次檢測細(xì)胞中EMT標(biāo)志物的表達(dá)情況.結(jié)果:與對照組比,TGF-β1處理72 h時能引起16HBE細(xì)胞發(fā)生EMT,細(xì)胞形態(tài)在72 h改變明顯,α-SMA和Vimentin表達(dá)增加,E-cadherin表達(dá)下降(P<0.01);轉(zhuǎn)染miR-152 mimics,細(xì)胞內(nèi)miR-152的表達(dá)顯著增加(P<0.01);與miR-NC組比,轉(zhuǎn)染miR-152過表達(dá)后,氣管上皮細(xì)胞中α-SMA和Vimentin的表達(dá)降低(P<0.01),E-cadherin表達(dá)增加(P<0.01),EMT受到抑制.結(jié)論:TGF-β1能誘導(dǎo)離體培養(yǎng)的氣管上皮細(xì)胞發(fā)生EMT;miR-152可以抑制TGF-β1誘導(dǎo)的氣管上皮細(xì)胞EMT的發(fā)生.

哮喘;氣管上皮細(xì)胞;間質(zhì)細(xì)胞;miR-152

哮喘是最常見的兒科疾病之一,其特征是嗜酸性粒細(xì)胞性氣道炎癥、可逆性氣道阻塞、氣道高反應(yīng)性和氣道重塑[1].大多數(shù)國家的哮喘患病率逐年上升,哮喘的治療需要花費(fèi)大量人力、物力和財力[2],但現(xiàn)有的控制哮喘的方式并不令人滿意.研究發(fā)現(xiàn)氣管上皮細(xì)胞向間質(zhì)細(xì)胞轉(zhuǎn)化(epithelialmesenchymal transition,EMT)在氣道重塑中具有重要的作用[3].轉(zhuǎn)化生長因子β1(transforming growth factor-β1,TGF-β1)是公認(rèn)的可以誘導(dǎo)EMT的因子,并且有報道稱哮喘患者肺組織內(nèi)TGF-β表達(dá)上調(diào)[4].因此,本研究采用10 ng/mL的TGF-β1來誘導(dǎo)氣管EMT發(fā)生.MicroRNA(miRNA)是一種非編碼蛋白的小分子RNA,大量研究證實(shí)miRNA在兒童哮喘診斷和治療中的作用[5-7].MiR-152是miR-148/152家族的一員,可作為腫瘤抑制因子參與腫瘤進(jìn)展.有研究發(fā)現(xiàn)miR-152的表達(dá)水平與哮喘的療效相關(guān)[8].

本研究通過qRT-PCR發(fā)現(xiàn)TGF-β1處理的氣管上皮細(xì)胞內(nèi)miR-152的表達(dá)水平下降,提示miR-152可能參與TGF-β1誘導(dǎo)的EMT.隨后進(jìn)一步通過轉(zhuǎn)染miR-152 mimics至人氣管上皮細(xì)胞,研究miR-152在TGF-β1誘導(dǎo)的氣管EMT中的作用,以期尋找早期干預(yù)哮喘氣道重塑的新途徑.

1 材料和方法

1.1 材料 人支氣管上皮細(xì)胞16HBE細(xì)胞株購自美國模式培養(yǎng)物集存庫(ATCC);TGF-β1抗體購自美國Sigma-Aldrich公司;MEM培養(yǎng)液、胰蛋白酶、1%青鏈霉素、胎牛血清購于美國Gibco公司;RNA提取試劑、轉(zhuǎn)染試劑脂質(zhì)體Lipofectamine2000購于美國Invitrogen公司;miR-152 mimics及陰性對照miR-NC、PCR相關(guān)引物由上海生工公司設(shè)計合成;Takara反轉(zhuǎn)錄試劑盒、SYBR Green I熒光染料試劑盒購于大連寶生物公司;兔或鼠抗人E-cadherin、Vimentin、α平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)、GAPDH多克隆抗體購自英國Abcam公司;Western blot相關(guān)試劑、辣根過氧化酶標(biāo)記的山羊抗兔或者抗鼠二抗購自江蘇碧云天生物技術(shù)研究所;倒置顯微鏡購自日本Olympus公司;ABI 7500 Fast PCR擴(kuò)增儀購自美國Applied Bio-systems公司.

1.2 方法

1.2.1 細(xì)胞培養(yǎng)和轉(zhuǎn)染:人支氣管上皮細(xì)胞16HBE在含有10%胎牛血清和1%青鏈霉素的MEM培養(yǎng)液中培養(yǎng),常規(guī)置于37 ℃、5% CO2環(huán)境下生長.細(xì)胞融合至90%左右時常規(guī)傳代.配置10 ng/mL的TGF-β1的溶液并無菌處理,細(xì)胞在接受藥物處理前先用無血清培養(yǎng)液饑餓處理24 h.在倒置顯微鏡下(200倍)觀察不同時間TGF-β1處理后細(xì)胞的形態(tài)學(xué)變化.miR-152 mimics轉(zhuǎn)染按照Invitrogen公司提供的轉(zhuǎn)染說明書,應(yīng)用Lipofectamine2000進(jìn)行轉(zhuǎn)染.

1.2.2 實(shí)驗分組:細(xì)胞分為無TGF-β1處理組(對照組)、TGF-β1處理24 h組、48 h組和72 h組;在miR-152 mimics轉(zhuǎn)染支氣管上皮細(xì)胞后,將細(xì)胞分為TGF-β1處理48 h組、TGF-β1+miR-NC組和TGF-β1+miR-152 mimics組.

1.2.3 qRT-PCR檢測基因表達(dá):用苯酚氯仿抽提法提取各組細(xì)胞中的總RNA,在NanoDrop2000紫外分光光度計測定A260 mm、A280 mm及RNA濃度.按照說明書將總RNA反轉(zhuǎn)錄成cDNA,反應(yīng)體系為20 μL,反應(yīng)條件為:16 ℃(30 min),45 ℃(30 min),80 ℃(5 min).隨后,以U6和GAPDH為內(nèi)參基因,運(yùn)用SYBR Green I法在ABI 7500 Fast PCR擴(kuò)增儀上檢測目的基因的表達(dá)情況,反應(yīng)條件為:預(yù)變性94 ℃(5 min),變性94 ℃(30 s),退火60 ℃(30 s),延伸72 ℃(60 s),擴(kuò)增40個循環(huán);最后72 ℃延伸8 min.每組樣品重復(fù)3次.目的基因的相對表達(dá)量采用2-ΔΔCt法計算.各引物序列見表1.

表1 qRT-PCR相關(guān)引物序列

1.2.4 Western blot檢測蛋白表達(dá)量:提取各組細(xì)胞總蛋白,用BCA法定量蛋白,SDS-PAGE每孔加入20 μL的樣品進(jìn)行蛋白電泳,隨后用孔徑0.45 μm的PVDF膜行轉(zhuǎn)膜.常溫下TBST洗膜3次、封閉1 h,孵育α-SMA抗體(1:1 000)、E-cadherin(1:500)、Vimentin(1:2 000)和GAPDH(1:5 000),4 ℃過夜,常溫下TBST洗膜3次,再加入辣根過氧化物酶標(biāo)記羊抗兔(鼠)二抗孵育2 h,ECL化學(xué)發(fā)光法顯影;柯達(dá)膠片暗室顯影,Quantity One 4.0軟件計算蛋白條帶灰度值.蛋白表達(dá)強(qiáng)度以各條帶灰度值/GAPDH灰度值表示.

1.3 統(tǒng)計學(xué)處理方法 采用SPSS20.0和GraphPad Prism 5.5軟件進(jìn)行統(tǒng)計分析.計量資料以表示,多組比較采用單因素方差分析和SNK-q檢驗.P<0.05為差異有統(tǒng)計學(xué)意義.

2 結(jié)果

2.1 TGF-β1刺激對氣管EMT的影響 對照組16HBE細(xì)胞分布呈鋪石路樣,形態(tài)成橢圓形或者是多邊,細(xì)胞間存在連接(見圖1A);TGF-β1 24 h組細(xì)胞形態(tài)改變不大(見圖1B),TGF-β1 72 h組細(xì)胞形態(tài)變?yōu)殚L梭形,并且細(xì)胞間隙較大(見圖1C).qRT-PCR檢測結(jié)果表明,與對照組比,TGF-β1 24 h組α-SMA和Vimentin mRNA開始增加,TGF-β1 72 h組α-SMA和Vimentin mRNA明顯升高,E-cadherin mRNA下降(P<0.01),見表2.Western blot發(fā)現(xiàn)16HBE細(xì)胞內(nèi)α-SMA、Vimentin和E-cadherin蛋白的表達(dá)量在TGF-β1刺激后呈現(xiàn)出和相應(yīng)的mRNA改變相同的趨勢(P<0.01),見圖2.

2.2 TGF-β1刺激氣管上皮細(xì)胞后miR-152的改變采用qRT-PCR發(fā)現(xiàn)10 ng/mL的TGF-β1處理16HBE細(xì)胞后,細(xì)胞內(nèi)miR-152的表達(dá)量呈下降趨勢,在24 h開始下降(P<0.01),72 h和24 h比差異無統(tǒng)計學(xué)意義(P>0.05),見表2.

圖1 TGF-β1刺激對氣管上皮細(xì)胞形態(tài)學(xué)的影響(X200)

圖2 TGF-β1刺激對氣管EMT相關(guān)蛋白的改變

2.3 miR-152對氣管EMT的影響 轉(zhuǎn)染miR-152 mimics后,對照組、TGF-β1組、TGF-β1+miR-NC組和TGF-β1+miR-152 mimics組16HBE細(xì)胞內(nèi)miR-152的2-△△CT值分別為1.03±0.13,0.62±0.06,0.67±0.10和31.70±1.31(P<0.01).qRT-PCR結(jié)果顯示,TGF-β1、TGF-β1+miR-NC和TGF-β1+miR-152 mimics組EMT標(biāo)志蛋白存在明顯差異;與TGF-β1+miR-NC組比,TGF-β1+miR-152 mimics組α-SMA和Vimentin mRNA下降,而E-cadherin mRNA表達(dá)升高(均P<0.01),見表3.Western blot檢測發(fā)現(xiàn),與TGF-β1組比,TGF-β1+miR-152 mimics組細(xì)胞中α-SMA和Vimentin蛋白表達(dá)減少,但E-cadherin蛋白表達(dá)增加(P<0.01),見圖3.

3 討論

目前治療哮喘的有關(guān)藥物,包括糖皮質(zhì)激素、白三烯拮抗劑以及長效β2受體激動劑,對阻止或逆轉(zhuǎn)氣道重塑的作用非常有限[9].氣道重塑的始動因素為氣管內(nèi)免疫細(xì)胞分泌炎癥介質(zhì),包括TGF-β、腫瘤壞死因子α和白介素等[10].有研究認(rèn)為由于兒童時期肺組織生長迅速,氣道重塑很可能在兒童哮喘患者中已經(jīng)發(fā)生[11].氣管EMT在哮喘相關(guān)的氣道重塑中具有重要作用,抑制EMT可以有效阻止氣道重塑的進(jìn)展[12].氣管上皮細(xì)胞可以通過EMT向肌成纖維細(xì)胞轉(zhuǎn)變,引起細(xì)胞收縮能力增加和氣道的高反應(yīng)性[3].EMT過程主要表現(xiàn)為氣管上皮細(xì)胞間連接缺失,變得松散;細(xì)胞極性消失;細(xì)胞形態(tài)由橢圓形向梭形轉(zhuǎn)變;上皮細(xì)胞標(biāo)志物細(xì)胞角蛋白、緊密連接蛋白和E-cadherin下降,而間質(zhì)細(xì)胞標(biāo)志物Vimentin和N-cadherin表達(dá)升高;肌成纖維細(xì)胞相關(guān)蛋白α-SMA、成纖維細(xì)胞特異性蛋白Collagen等表達(dá)增加[13].TGF-β已被證實(shí)可以誘導(dǎo)內(nèi)皮細(xì)胞EMT,是目前最常用的用來誘導(dǎo)EMT的藥物,因此本研究采用10 ng/mL的TGF-β1來誘導(dǎo)離體培養(yǎng)的氣管上皮16HBE發(fā)生EMT[14].

表2 TGF-β1刺激后α-SMA、Vimentin,E-cadherin mRNA和miR-152的改變(n=3,

表2 TGF-β1刺激后α-SMA、Vimentin,E-cadherin mRNA和miR-152的改變(n=3,

與對照組比:aP<0.01;與TGF-β1 24 h組比:bP<0.01

組別 α-SMA Vimentin E-cadherin miR-152對照組 0.99±0.09 0.96±0.04 0.98±0.14 1.09±0.11 TGF-β1 24 h組 1.35±0.13a 1.24±0.08a 0.81±0.09a 0.69±0.06a TGF-β1 72 h組 1.91±0.08b 1.55±0.14b 0.55±0.10b 0.52±0.09 F 36.78 87.96 36.40 24.61 P<0.001 <0.001 <0.001 0.0013

表3 MiR-152過表達(dá)對TGF-β1誘導(dǎo)的16HBE細(xì)胞EMT相關(guān)基因的影響(n=3,

表3 MiR-152過表達(dá)對TGF-β1誘導(dǎo)的16HBE細(xì)胞EMT相關(guān)基因的影響(n=3,

與TGF-β1+miR-NC組比:aP<0.01

組別 α-SMA Vimentin E-cadherin TGF-β1組 1.79±0.12 1.48±0.17 0.62±0.06 TGF-β1+miR-NC組 1.75±0.16 1.39±0.14 0.65±0.11 TGF-β1+miR-152 mimics組 0.96±0.08a 1.01±0.05a 0.95±0.06a F 39.32 137.43 26.44 P<0.001 <0.001 0.0019

圖3 MiR-152過表達(dá)對TGF-β1誘導(dǎo)的16HBE細(xì)胞EMT相關(guān)蛋白的影響

TGF-β1處理16HBE細(xì)胞24 h和48 h,通過細(xì)胞大體形態(tài)、EMT標(biāo)志物mRNA和蛋白的改變等綜合表現(xiàn),驗證了10 ng/mL TGF-β1誘導(dǎo)實(shí)驗細(xì)胞EMT的有效性.哮喘患者氣管上皮細(xì)胞內(nèi)miRNAs表達(dá)存在異常,提示其可能參與上皮細(xì)胞異常介導(dǎo)的哮喘病理生理機(jī)制[15].因此,我們檢測TGF-β1處理的細(xì)胞內(nèi)miR-152的表達(dá)情況,意外發(fā)現(xiàn)其表達(dá)下降,在設(shè)置時間梯度后發(fā)現(xiàn)miR-152在TGF-β1處理24 h后下降明顯,而72 h表達(dá)量與前者差異無統(tǒng)計學(xué)意義.鑒于大量研究發(fā)現(xiàn)miRNA在內(nèi)皮細(xì)胞EMT中的作用[16-17],我們猜測miR-152也參與了TGF-β1誘導(dǎo)的氣管EMT.

研究表明miR-152作為一個抑癌因子參與腫瘤的發(fā)生發(fā)展[18],但是對于其在哮喘中的作用的研究卻寥寥無幾.2015年,NAIDOO等[8]在他汀類藥物改善哮喘的機(jī)制研究中,使用辛伐他汀處理B淋巴母細(xì)胞永生細(xì)胞株,檢測miR-152家族(包括miR-148a、miR-148b和miR-152)的表達(dá)變化,發(fā)現(xiàn)miR-152表達(dá)明顯升高,并且上調(diào)的miR-152能靶向結(jié)合HLA-G的3'-UTR,引起其降解,從而改善肺功能.為了探討miR-152是否參與哮喘的氣道重塑,我們通過miRNA模擬物轉(zhuǎn)染提高細(xì)胞內(nèi)miR-152的表達(dá)量,結(jié)果發(fā)現(xiàn)miR-152過表達(dá)可以抑制TGF-β1誘導(dǎo)的16HBE細(xì)胞的EMT.并且miR-152過表達(dá)不僅逆轉(zhuǎn)EMT相關(guān)蛋白,同時對EMT相關(guān)標(biāo)志物轉(zhuǎn)錄層面產(chǎn)生影響,這提示miR-152很可能通過作用于某個靶基因從而調(diào)節(jié)這些相關(guān)基因的轉(zhuǎn)錄.因此,在后續(xù)實(shí)驗中我們將進(jìn)一步尋找miR-152作用的可能潛在靶點(diǎn),深入探討miR-152在TGF-β1誘導(dǎo)氣管上皮細(xì)胞EMT中的作用,并通過動物實(shí)驗進(jìn)一步證實(shí)miR-152參與哮喘的氣道重塑.

綜上所述,本研究通過細(xì)胞實(shí)驗發(fā)現(xiàn)miR-152介導(dǎo)TGF-β1誘導(dǎo)的氣管EMT,為抑制EMT相關(guān)的哮喘氣道重塑治療提供新的思路.

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(本文編輯:趙翠翠)

The effects of miR-152 on transforming growth factor-beta1-mediated epithelial-mesenchymal transition in human bronchial epithelial cells

ZHU Ronghe, SUN Yuanyuan, QIAN Yan.
Department of Pediatrics, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015

Objective:To verify whether transforming growth factor-beta1 (TGF-β1) could induce epithelial-mesenchymal transition (EMT) in vitro and to determine the role of miR-152 in TGF-β1-mediated EMT in bronchial epithelial cells.Methods:10 ng/mL of TGF-β1 was used to induce the EMT of human bronchial epithelial cells 16HBE. Morphological changes were observed and qRT-PCR and Western blot were employed to test the alteration of αSMA, E-cadherin and Vimentin, thus to verify the occurrence of EMT. qRT-PCR was then used to test the expression profile of miR-152 in TGF-β1 treated 16HBE cells. After miR-152 was successfully transfected into 16HBE cells, EMT related markers were then detected using qRT-PCR and Western blot.Results:Compared with control group, TGF-β1-treated cells presented a myofibroblast-like morphology,especially at 72 h, characteristic by the loss of cell-to-cell junction, denovo expression of α-SMA and Vimentin,and loss of epithelial marker E-cadherin (P<0.01). The expression levels of miR-152 in TGF-β1 treated 16HBE cells were dramatically increased after transfected with miR-152 mimics (P<0.01). Compared with the control group, α-SMA and Vimentin were decreased, whereas E-cadherin was increased in the miR-152 group (P<0.01),suggesting overexpression of miR-152 could reverse the EMT induced by TGF-β1 in 16HBE cells.Conclusion:TGF-β1 induces EMT of human bronchial epithelial cells in vitro; miR-152 inhibits TGF-β1-mediated EMT in 16HBE cells.

asthma; bronchial epithelial cells; mesenchymal cells; miR-152

R725.6

A

10.3969/j.issn.2095-9400.2017.10.008

2017-04-13

溫州市科技計劃項目(2014Y0143).

朱融和(1985-),女,浙江溫州人,住院醫(yī)師,碩士.

錢燕,主任醫(yī)師,碩士生導(dǎo)師,Email:qianyan_paw@163.com.