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microRNA-34a體外調(diào)控鼻咽癌鱗狀腫瘤干細(xì)胞表型

2016-10-13 07:16江文靜蔣學(xué)范胡未鳴
中國現(xiàn)代醫(yī)生 2016年21期
關(guān)鍵詞:擬態(tài)原代鱗狀

江文靜 蔣學(xué)范 胡未鳴

浙江省人民醫(yī)院耳鼻喉科,浙江杭州310000

microRNA-34a體外調(diào)控鼻咽癌鱗狀腫瘤干細(xì)胞表型

江文靜蔣學(xué)范胡未鳴▲

浙江省人民醫(yī)院耳鼻喉科,浙江杭州310000

目的探討microRNA-34a(miR-34a)對鼻咽癌鱗狀腫瘤干細(xì)胞表型的體外調(diào)控作用。方法利用原代培養(yǎng)的人類鼻咽癌細(xì)胞,采用流式分選法定量并收集ALDH(aldehyde dehydrogenase)+腫瘤干細(xì)胞。通過RT-qPCR分析miR-34與腫瘤干細(xì)胞相關(guān)因子(Sox2、Nanog、Oct3/4)在ALDH+和ALDH-細(xì)胞中的表達(dá)情況。進(jìn)一步用miR-34a擬態(tài)轉(zhuǎn)染評估其對鼻咽癌腫瘤干細(xì)胞標(biāo)記物以及相關(guān)因子的表達(dá)調(diào)控能力。結(jié)果原代鼻咽癌細(xì)胞中表達(dá)ALDH的細(xì)胞約占15.43%左右。與ALDH-細(xì)胞相比,miR-34a表達(dá)水平在大多數(shù)ALDH+細(xì)胞中顯著下調(diào)(-13.2 vs-4.1倍),3種腫瘤干細(xì)胞相關(guān)因子表達(dá)顯著增加(最高36.8倍)。ALDH+細(xì)胞轉(zhuǎn)染miR-34a擬態(tài)24 h、48 h、72 h后miR-34a mRNA水平顯著增加,48 h時(shí)達(dá)峰值水平(4.49倍,P<0.01),而3種腫瘤干細(xì)胞相關(guān)因子表達(dá)水平顯著降低。結(jié)論恢復(fù)miR-34a表達(dá)顯著抑制人類原代鼻咽癌干細(xì)胞表型的形成。調(diào)控鼻咽癌和腫瘤干細(xì)胞中miR-34a的表達(dá)可能降低腫瘤治療后的轉(zhuǎn)移和復(fù)發(fā)率。

鼻咽癌;腫瘤干細(xì)胞;microRNA-34a;轉(zhuǎn)染;擬態(tài);醛脫氫酶

[Abstract]Objective To investigate the role of miR-34a in regulating cancer stem cell(CSC)phenotype of nasopharyngeal squamous cell carcinoma in vitro.Methods The cells were isolated from patients with nasopharyngeal carcinoma(NPC)and cultured in vitro.Flow-cytometry was used to quantify and enrich for ALDH+cancer stem cells(CSCs).RT-qPCR was performed to analyze expression patterns of miR-34a and CSC-related transcription factors(CSC-TFs)(Sox2,Nanog,Oct3/4)for ALDH+and ALDH-cells.Transfection of miR-34a mimics was used to evaluate its regulatory potential for CSC marker profiles as well as CSC-TFs expression in NPC-CSC.Results The expression of ALDH was found in around 15.43%of primary NPC cells.miR-34a expression levels were significantly downregulated in the majority of ALDH+cells derived from primary NPC as compared to ALDH-cells(-13.2 vs-4.1 fold).For CSC-related TF expression,ALDH+cells showed a significantly increased level compared to ALDH-cells(up to 36.8 fold).miR-34a mRNA level in ALDH+cells after transfection with miR-34a mimics significantly increased after 24 h,48 h and 72 h,and the peak of miR-34a level was found 48 h post-transfection(4.49 fold,P<0.01).Transfection of miR-34a mimics significantly reduced the CSC-related TF expression level in ALDH+cells.Conclusion Restoration miR-34a significantly inhibited the formation of CSC-phenotype in human primary NPC cells.Therapeutic modulation of miR-34a in NPC and CSCs may reduce the rate of metastasis and recurrence of tumors after therapy.

[Key words]Nasopharyngeal carcinoma;Cancer stem cells;microRNA-34a;Transfection;Mimics;Aldehyde dehydrogenase

近年來,通路靶向抑制治療鼻咽癌(nasopharyngeal carcinoma,NPC)的報(bào)道已取得了一些進(jìn)展,但其研究僅限于NPC細(xì)胞株,未充分考慮到腫瘤細(xì)胞株在逐代的培養(yǎng)過程中可能會(huì)發(fā)生某些生物學(xué)行為的改變,如細(xì)胞活性、藥物敏感性等等[1]。因此,為使研究更貼近臨床人體內(nèi)實(shí)驗(yàn),本文探討了miR-34a和人類原代NPC干細(xì)胞生物學(xué)行為之間的關(guān)系。本研究首先采用流式分選法分選出ALDH+和ALDH-細(xì)胞,然后通過實(shí)時(shí)定量PCR檢測兩種細(xì)胞中CSCs相關(guān)的3種轉(zhuǎn)錄因子(CSC-TFs:Sox2、Nanog、Oct3/4)[2]及miR-34a的表達(dá)是否有差異,并將ALDH+細(xì)胞轉(zhuǎn)染miR-34a擬態(tài),比較不同作用時(shí)間(24 h、48 h、72 h)后miR-34a及3種轉(zhuǎn)錄因子表達(dá)量的變化,探討miR-34a轉(zhuǎn)染對NPC干細(xì)胞增殖是否有抑制效應(yīng)。

1 材料與方法

1.1鼻咽癌原代細(xì)胞培養(yǎng)

新鮮鼻咽鱗狀細(xì)胞癌組織由本院耳鼻喉科提供。本研究經(jīng)患者知情同意和醫(yī)院倫理委員會(huì)審查通過,研究工作從2015年8月開展至今。步驟:①適量新鮮標(biāo)本置于15 mL離心管中,裝2 mL含10℅FBS(Gibco公司)的RPMI-1640(Gibco公司)培養(yǎng)基。②去除外周壞死組織,無血清培養(yǎng)基充分漂洗3次。③將組織塊剪成0.5 mm大小,加入適量0.25℅胰酶,37℃消化8~10 min;含10℅FBS的培養(yǎng)基終止消化,吹打均勻后200目篩網(wǎng)過濾;將濾液接種于25 cm2培養(yǎng)瓶中,培養(yǎng)基3 mL,置于37℃、5%CO2、濕潤空氣的密閉式培養(yǎng)箱中培養(yǎng)。④96孔板單細(xì)胞克隆分離提純細(xì)胞。

1.2Aldefluor測定和FACS分選

Aldefluor測定試劑盒(StemCell公司)檢測細(xì)胞活性;將細(xì)胞用胰蛋白酶消化制成單細(xì)胞懸液,不含Ca2+/Mg2+的PBS洗滌,重懸浮于含5 μL ALDH底物的1 mL Aldefluor緩沖液中(1×106個(gè)細(xì)胞/mL),暗室37℃孵育30~40 min。加入ALDH抑制劑(DEAB,50 mmol/L)作為陰性對照。所有樣本使用250×g離心5 min,棄上清。緩沖液洗滌細(xì)胞兩次后加入ALDH緩沖液,并置于冰上待用。將細(xì)胞懸浮于PBS緩沖中,107個(gè)細(xì)胞/mL,應(yīng)用流式細(xì)胞儀Aria cell sorter進(jìn)行分選(BD Biosciences公司)。

1.3實(shí)時(shí)定量PCR

Trizol試劑(Gibco公司)分離總RNA,Poly A尾和cDNA使用Ncode VILO miR cDNA(Invitrogen)合成。使用Chromo 4(BioRad)PRC儀及SYBR Green ERTM qPCR SuperMix試劑(Invitrogen公司)進(jìn)行RT-qPCR分析。參照基因GAPDH。啟動(dòng)序列見表1。

1.4microRNAs(miRs)的轉(zhuǎn)染

為轉(zhuǎn)染miR擬態(tài)(mimics),首先將ALDH+細(xì)胞制成單細(xì)胞懸液,然后接種于含完全培養(yǎng)基的6孔板中,密度8×104個(gè)細(xì)胞/孔。50 nmol/L miR-34a擬態(tài)(mimics)轉(zhuǎn)染細(xì)胞,陰性對照(NC)或空白對照(MOCK)用Lipofectamine RNAiMAX試劑(Invitrogen),且不含抗生素Opti-MEM(Invitrogen)。使用BLOCK-ITTM公司的Alexa Fluor紅色熒光指示劑處理后,通過熒光顯微鏡對轉(zhuǎn)染的細(xì)胞進(jìn)行細(xì)胞計(jì)數(shù)以確定轉(zhuǎn)染效率。RT-qPCR評估對ALDH+細(xì)胞轉(zhuǎn)染前后miR-34a的表達(dá)及3種CSC-TFs的表達(dá)進(jìn)行定量,后者分別在轉(zhuǎn)染后的24 h、48 h和72 h測定。

1.5統(tǒng)計(jì)學(xué)方法

數(shù)據(jù)用2△△Ct進(jìn)行統(tǒng)計(jì)分析[3],相關(guān)CT值見表2~5。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1ALDH在NPC中表達(dá)情況

流式分選ALDH+腫瘤干細(xì)胞,見圖1。DEAB抑制下分采用流式分選法定性并分選ALDH+細(xì)胞。圖1左顯示DEAB抑制下ALDH+細(xì)胞分選情況,圖1右顯示無DEAB抑制劑下ALDH+細(xì)胞分選情況,R2區(qū)域顯示ALDH+細(xì)胞熒光強(qiáng)度(原代鼻咽癌細(xì)胞中表達(dá)ALDH的細(xì)胞均占15.43%左右)。

2.2RT-qPCR分析CSC相關(guān)轉(zhuǎn)錄因子(CSC-TFs: Sox2、Nanog、Oct3/4)mRNA及miR-34a的表達(dá)

圖1 DEAB抑制下流式分選ALDH+和ALDH-細(xì)胞

RT-qPCR分析3種CSC-TFs的表達(dá),ALDH+細(xì)胞中3種CSC-TFs的表達(dá)均顯著高于ALDH-細(xì)胞(最高36.8倍)(P<0.05或P<0.01),見圖2、表2;RT-qPCR分析miR-34a的表達(dá),ALDH+細(xì)胞中miR-34a表達(dá)量顯著低于ALDH-細(xì)胞(-13.2 vs-4.1倍),見圖3、表3。

表1 RT-PCR引物序列(5’→3’)

2.3miR-34a超表達(dá)降低ALDH+細(xì)胞的干細(xì)胞特性

ALDH+細(xì)胞轉(zhuǎn)染前后miR-34a表達(dá)量比較,轉(zhuǎn)染后24 h、48 h、72 h其表達(dá)量顯著增加,其中48 h時(shí)表現(xiàn)更明顯(4.49倍),然而與對照組比較,Mock組轉(zhuǎn)染前后miR-34a表達(dá)量無明顯統(tǒng)計(jì)學(xué)差異,見圖4、表4。ALDH+細(xì)胞轉(zhuǎn)染后CSC相關(guān)轉(zhuǎn)錄因子(Nanog、Oct3/4)表達(dá)顯著降低,Sox2表達(dá)無明顯統(tǒng)計(jì)學(xué)差異,見圖5、表5。

圖2 ALDH+細(xì)胞中CSC相關(guān)轉(zhuǎn)錄因子(CSC-TFs:Sox2]Nanog]Oct3/4)的表達(dá)顯著高于ALDH-細(xì)胞,*P<0.05,**P<0.01

圖3 ALDH+細(xì)胞中miR-34a表達(dá)量顯著低于ALDH-細(xì)胞,***P<0.001

表2 ALDH+和ALDH-細(xì)胞中CSC相關(guān)轉(zhuǎn)錄因子表達(dá)CT值

表3 ALDH+和ALDH-細(xì)胞miR-34a表達(dá)CT值

表4 miR-34a在3組ALDH+細(xì)胞且不同時(shí)間內(nèi)表達(dá)的CT值

圖4 ALDH+細(xì)胞轉(zhuǎn)染前后miR-34a表達(dá)量比較,轉(zhuǎn)染后24 h]48 h]72 h其表達(dá)量顯著增加,其中48 h時(shí)表現(xiàn)更為明顯,*P<0.05,**P<0.01

圖5 ALDH+細(xì)胞轉(zhuǎn)染后CSC相關(guān)轉(zhuǎn)錄因子(Nanog,Oct3/4)表達(dá)顯著降低,Sox2表達(dá)無明顯統(tǒng)計(jì)學(xué)差異,*P<0.05,**P<0.01

3 討論

CSCs是腫瘤組織細(xì)胞的一小部分,它們具有無限的增殖潛能[4]。同時(shí),CSCs是腫瘤細(xì)胞真正的“種子”,在某些腫瘤中表現(xiàn)出重要的生物學(xué)特性[5],它們與腫瘤的發(fā)生、致癌性、轉(zhuǎn)移和復(fù)發(fā)等密切相關(guān),并對傳統(tǒng)的放化療具有抵抗性[6-7]。Ginestier等[8]發(fā)現(xiàn)將人類乳腺癌ALDH+細(xì)胞接種于非肥胖糖尿病/重癥聯(lián)合免疫缺陷的小鼠體內(nèi)能形成腫瘤,因此推測這些細(xì)胞可能包含CSCs。隨后,利用ALDH1活性能從乳腺癌中鑒別并分離出CSCs[9]。ALDH在人類頭頸部鱗狀細(xì)胞癌細(xì)胞株中分離出的CSC中也呈現(xiàn)高表達(dá)[10]。本研究中我們采用流式分選獲取具有CSC表型的ALDH+和ALDH-細(xì)胞,進(jìn)一步證實(shí)了ALDH+細(xì)胞中3種CSCTFs的表達(dá)均顯著高于ALDH-細(xì)胞。

microRNAs(miRs)是非編碼短單鏈RNAs,在正常和腫瘤細(xì)胞中調(diào)控目的mRNAs基因的翻譯,頻繁失調(diào)能促進(jìn)腫瘤進(jìn)展。miR-34a屬于腫瘤抑制因子,它直接作用于p53轉(zhuǎn)錄后水平。在p53缺陷的人類胰腺癌細(xì)胞中,miR-34a過度表達(dá)抑制細(xì)胞增殖,細(xì)胞周期進(jìn)展及自我更新,表明miR-34a可能恢復(fù)p53功能,直接作用于與CSC分化和自我更新相關(guān)的下游靶基因[11]。MiR-34a也能抑制前列腺癌[12]和乳腺癌[13]腫瘤干細(xì)胞相關(guān)特性和功能的表達(dá)。最新研究顯示,miR-34a調(diào)節(jié)結(jié)腸癌干細(xì)胞的不對稱分裂,從而促進(jìn)腫瘤生長[14]。此外,miR-34a不僅抑制非小細(xì)胞肺癌H1299細(xì)胞株的增殖,并能促進(jìn)其凋亡[15]。越來越多的研究表明,針對miRNA靶向抑制CSCs在殺滅腫瘤細(xì)胞的同時(shí)也能預(yù)防腫瘤復(fù)發(fā)[16]。本研究也證實(shí)miR-34a在ALDH+細(xì)胞中表達(dá)水平顯著低于ALDH-細(xì)胞,同時(shí)miR-34a能抑制原代NPC中ALDH+細(xì)胞相關(guān)轉(zhuǎn)錄因子的表達(dá)。近來相關(guān)研究也表明miR-34a在頭頸部CSC中下調(diào)可能誘發(fā)腫瘤生長和發(fā)生[17]。腫瘤干細(xì)胞相關(guān)因子Nanog、Oct3/4、Sox2在miRNA誘導(dǎo)的結(jié)腸癌細(xì)胞中均呈現(xiàn)高表達(dá)[18]。然而,在我們的研究中miR-34a擬態(tài)轉(zhuǎn)染在增加miR-34a表達(dá)的同時(shí),只減少ALDH+細(xì)胞中相關(guān)CSC表型Nanog、Oct3/4的表達(dá),而Sox2表達(dá)無明顯統(tǒng)計(jì)學(xué)差異。

本研究顯示原代NPC細(xì)胞中含ALDH+細(xì)胞,即NPC干細(xì)胞。RT-qPCR分析結(jié)果顯示ALDH+細(xì)胞中3種CSC-TFs的表達(dá)均顯著高于ALDH-細(xì)胞,但miR-34a表達(dá)量顯著低于ALDH-細(xì)胞。由此推斷,miR-34a的表達(dá)在NPC干細(xì)胞中明顯少于非NPC干細(xì)胞。進(jìn)一步對分選出的ALDH+細(xì)胞進(jìn)行miR-34a擬態(tài)轉(zhuǎn)染,結(jié)果顯示轉(zhuǎn)染24 h、48 h、72 h后miR-34a表達(dá)量顯著增加,其中48 h時(shí)表現(xiàn)更明顯,然而對照組無明顯統(tǒng)計(jì)學(xué)差異,表明miR-34a表達(dá)呈一定時(shí)間依賴性。同時(shí)分別對ALDH+細(xì)胞轉(zhuǎn)染后3種CSC-TFs的表達(dá)進(jìn)行分析,發(fā)現(xiàn)轉(zhuǎn)染后CSC相關(guān)表達(dá)因子Nanog、Oct3/4的表達(dá)顯著降低,而Sox2表達(dá)無明顯統(tǒng)計(jì)學(xué)差異。據(jù)此研究表明,miR-34a體外抑制鼻咽癌鱗狀腫瘤干細(xì)胞表型的表達(dá)。因此,使用針對相關(guān)通路靶向提高miR-34a表達(dá)從而抑制CSC的生長,將可能成為徹底清除人類腫瘤包括NPC在內(nèi)的一個(gè)革新性治療策略。

此外,研究發(fā)現(xiàn)在口咽部鱗狀細(xì)胞癌中,人乳頭瘤病毒(human papillomavirus,HPV)是否感染對細(xì)胞生物學(xué)和臨床特性也起到一定作用,與HPV-DNA-細(xì)胞相比,HPV-DNA+腫瘤細(xì)胞中ALDH1A1的表達(dá)水平降低[19]。ALDH1A1作為一種腫瘤干細(xì)胞標(biāo)記之一,與頭頸部鱗狀細(xì)胞癌的預(yù)后密切相關(guān)[20]。因此,在以后的研究中,圍繞HPV感染及治療將待進(jìn)一步研究。

表5 ALDH+細(xì)胞中CSC相關(guān)轉(zhuǎn)錄因子在3組條件下表達(dá)的CT值

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microRNA-34a regulates cancer stem cell phenotype of squamous nasopharyngeal carcinoma in vitro

JIANG WenjingJIANG XuefanHU Weiming
Department of Otorhinolaryngology,Zhejiang Provincial People's Hospital,Hangzhou310000,China

R76

A

1673-9701(2016)21-0028-05

2016-05-07)

浙江省醫(yī)藥衛(wèi)生科技計(jì)劃項(xiàng)目(2016KYB024)▲

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