微RNA通過調節(jié)上皮間質轉化影響腫瘤轉移

楊麗華,沈星凱,李靜秋,楊杰,樂燕萍,龔朝輝

1. 寧波大學醫(yī)學院生物化學與分子生物學研究所,寧波 315211;

2. 浙江省病理生理學技術研究重點實驗室,寧波 315211

據(jù)估計,2013年僅美國就有580350人死于癌癥,平均每天約1600人,癌癥已成為僅次于心臟疾病的第二大死因[1]。在我國,根據(jù)中國衛(wèi)生統(tǒng)計年鑒(2012 版)統(tǒng)計,1973～1975、1990～1992、2004～2005年中國惡性腫瘤死亡抽樣回顧調查結果顯示,惡性腫瘤死亡率呈顯著上升趨勢,分別為 83.65/100000、108.26/100000和134.80/100000。癌癥致人死亡的原因近 90%是癌癥發(fā)生了轉移。轉移是惡性腫瘤最顯著的特征之一,它是一個多步驟的過程[2]。腫瘤細胞發(fā)生轉移的初始會發(fā)生上皮間質轉化(Epithelialmesenchymal transition,EMT),EMT過程又受到多種因素調控,如轉錄因子、微RNA(MicroRNA,miRNA)等。miRNA是一類內源性非編碼小分子RNA,與細胞生長、凋亡和信號轉導密切相關。同時,miRNA可以調控EMT過程,在腫瘤轉移發(fā)面發(fā)揮了重要作用。

1 EMT和腫瘤轉移

EMT是胚胎正常發(fā)育、傷口愈合和惡性上皮腫瘤發(fā)生過程中的一個極其重要的基本過程[3],它可以使上皮細胞發(fā)生形態(tài)改變,轉換成有運動能力的間充質細胞[4]。這些改變會使細胞-細胞之間和細胞-細胞外基質之間的連接受到破壞,因而細胞可以遷移到身體的其他部位參與組織重建和修復[5]。EMT在腫瘤細胞的遷移/侵襲以及多種癌癥進程方面也起到非常重要的作用,是腫瘤細胞轉移中的一個關鍵步驟,也是一個特定的信號通路[6,7]。EMT的特點是細胞粘附能力喪失,E-鈣粘蛋白(E-cadherin,CDH1)表達下降,間葉標志物(包括 N-鈣粘蛋白(N-cadherin)、波形蛋白(Vimentin)和纖維連接蛋白(Fibronectin)表達上升,細胞運動能力和侵襲能力增強[8]。原發(fā)部位的腫瘤細胞可以發(fā)生EMT來獲得運動能力和侵襲能力,而間質上皮轉化(Mesenchymalepithelial transition,MET)在腫瘤轉移的最后一步起到重要作用,即從血管或淋巴管中滲出的腫瘤細胞會恢復到上皮細胞狀態(tài),然后增殖形成巨大的甚至肉眼可見的繼發(fā)性腫瘤[9]。腫瘤EMT過程受內在因素和外在因素或者是二者共同作用的影響(圖1),其中內在因素包括腫瘤發(fā)生過程中產生的基因突變。突變的K-RAS途徑即是一個典型的例子[10],它可以單獨誘導 EMT,也可以聯(lián)合 EMT誘導因子如轉化生長因子-β(Transforming growth factor-β,TGF-β)或者缺氧誘導因子 -2α(Hypoxia-inducible factor-2α,HIF-2α)共同作用。在某種程度上,由煙草(Nicotiana tabacum L.)引起的缺氧或者炎性腫瘤微環(huán)境,通過細胞外信號通路的失調,創(chuàng)造一個促進EMT發(fā)生的環(huán)境,特別是 TGF-β和環(huán)氧化酶/前列腺素 E2途徑在腫瘤微環(huán)境中具有重要作用[11]。在肺癌中,缺氧腫瘤微環(huán)境誘導EMT主要是上調了堿性螺旋-環(huán)-螺旋類(Basic helix-loop helix,bHLH)轉錄因子和HIF的表達。在EMT的細胞內外信號通路中,EMT誘導轉錄因子和 miRNA起主要作用。在多發(fā)性腫瘤中,EMT被認為是轉移的標志,與許多轉錄因子緊密相關。已有研究表明,轉錄因子能夠促進 EMT和腫瘤轉移[12～16]。轉移過程中的多個步驟都受到這些轉錄因子調節(jié),而這些因子本身的表達同時受到miRNA的精細調控。

圖1 癌變過程中多種因素共同誘導EMT

2 影響EMT過程的轉錄因子

影響 EMT的轉錄抑制因子包括 Snail1[12]、Snail2[13]、Twist1[14]、Zeb1[15]、Zeb2[16]、Goosecoid[17]、FOXC2[18]、FoxQ1[19]、KLF8[20]、Prrx1[21]等。本文主要總結了 Snail、Twist和 Zeb轉錄因子家族及其在EMT和腫瘤轉移方面的網絡調控作用。

2.1 Snail鋅指轉錄抑制因子家族

鋅指簇可以與靶基因調控區(qū)的 E-box結合進行調控,Snail家族是EMT轉錄抑制因子中研究最多的。諸多研究表明,很多信號通路會誘導 Snail1和Snail2 的表達,包括 TGF-β[22]、Notch[23]和 Wnt[24]信號通路,活性氧[25]和缺氧[26]。其中,TGF-β信號通路是最重要的EMT誘導細胞因子之一,它可以激活包括Snail1/2在內的一系列EMT轉錄因子[27,28]。肝纖維化模型在缺氧條件下,基質細胞會分泌大量的TGF-β,隨后腫瘤細胞受到 TGF-β的刺激后,促進Snail1表達,誘發(fā)EMT[29],這表明EMT誘導的各種信號通路之間互有關聯(lián)。

2.2 Twist bHLH轉錄因子家族

參與EMT調控的bHLH轉錄因子包括E12和E47(E2A基因選擇性剪切產物)、Twist1和2、Id1-4[30],均可以直接負調控CDH1的表達。Twist1和Twist2表達異常誘發(fā) EMT的發(fā)生[14,31]。這是由于 Twist1可以結合Snail2啟動子區(qū),從而促進Snail2基因轉錄,過表達的 Snail2蛋白介導乳腺上皮腫瘤細胞發(fā)生EMT[32]。同時,Twist1亦可通過直接與血小板衍生生長因子受體α(Platelet-derived growth factor receptor-alpha,PDGFRα)啟動子區(qū)結合,促進 PDGFα及其下游基因的表達,促進侵襲性偽足的形成,引起腫瘤細胞的侵襲和轉移[33]。在干細胞和癌癥中經常呈現(xiàn)高表達的多梳抑制復合體蛋白 Bmi1同Twist1形成復合體,進而抑制CDH1和p16INK4a基因表達[34]。Twist亦可誘導miR-10b的表達,miR- 10b通過靶向抑制Homebox D10的翻譯,造成促轉移基因RHOC的表達上調,最終影響乳腺癌轉移[35]。一些癌基因可以誘導細胞異常增殖和異常轉化,但細胞衰老會抑制腫瘤發(fā)生EMT和轉移[36]。Twist2亦可下調CDH1的表達,參與TGF-β和p21誘導結腸癌細胞發(fā)生EMT這一過程[37]。在轉錄水平,Twist1主要受缺氧信號通路的調控[38]。在轉錄后水平,幾種微RNA可以直接靶向Twist1 mRNA[39]。此外,通過激酶作用,Twist1蛋白在 Ser68處發(fā)生磷酸化,使Twist1更穩(wěn)定,并且促進乳腺癌細胞的侵襲[40]。以上研究表明,Twist bHLH家族通過EMT影響腫瘤轉移。

2.3 Zeb鋅指轉錄因子家族

Zeb家族包括鋅指/同源結構域蛋白-ZEB1和ZEB2,在物種間高度保守,和其他轉錄因子共同作用調節(jié)EMT,并且在這一過程中受SUMO化、乙?；土姿峄恼{控。這些蛋白均可通過抑制上皮型標志物和激活間質型標志物的表達來誘發(fā) EMT過程。多種因子和信號通路可直接激活Zeb家族的表達,如生長激素和類固醇激素、缺氧條件下的HIF-1α、炎性細胞因子、配體(成纖維細胞生長因子、胰島素樣生長因子-1、血小板衍生生長因子等受體的配體)、Ras-ERK2-Fra1、NF-κB 和 JAK/STAT3 等腫瘤中經常處于激活狀態(tài)的下游通路、Wnt和Notch信號通路。Zeb家族也受miR-200的抑制,它們之間存在一個負反饋環(huán)路,最終誘發(fā)多種腫瘤的EMT[41,42]。

3 miRNA通過EMT調節(jié)腫瘤轉移

miRNA是一類長約 22 nt的小分子RNA,可在轉錄后水平通過結合靶 mRNA的 3′UTR阻止靶mRNA的翻譯或者促進靶mRNA的降解,實現(xiàn)對其靶基因的負調控[43]。據(jù)估計,miRNA可以調控人類30%左右蛋白的表達[44]。miRNA通過調節(jié)腫瘤細胞EMT相關靶蛋白表達,進而抑制或促進腫瘤轉移(表 1)。

3.1 miRNA通過 CDH1、波形蛋白和轉錄因子調節(jié)EMT

CDH1是 EMT/MET中非常重要的調控基因,miRNA可以通過多種機制對其進行調節(jié)。miR-9通過直接靶向CDH1 mRNA引發(fā)EMT,促進乳腺癌轉移。和未發(fā)生轉移的乳腺癌患者相比,發(fā)生轉移患者的原位癌組織中miR-9的表達升高。進一步研究發(fā)現(xiàn),miR-9增加細胞的運動和侵襲能力,因而使非轉移的乳腺癌細胞形成微轉移[46]。miR-23a也直接靶向CDH1調控肺癌發(fā)生EMT[49]。Xu等[45]研究表明,miR-25在食管鱗狀細胞癌中表達上調,其表達和淋巴結轉移及腫瘤分期相關,這是由于miR-25直接負調控CDH1,進而促進細胞侵襲和遷移。

表1 參與腫瘤EMT的miRNA

miRNA除了直接靶向CDH1外,亦可間接調控CDH1。miR-138表達下調會促進 EMT的發(fā)生,其直接靶點是波形蛋白[47]。除了波形蛋白,miR-138還靶向EMT相關的其他靶基因,如ZEB2和靶向zeste基因增強子人類同源物2 (Enhancer of zeste homologue 2,EZH2)。由于EZH2可抑制組蛋白去乙?；?/2(Histone deacetylases 1/2,HDAC1/2)和Snail的表達,間接降低 CDH1的表達,最終誘發(fā)鼻咽癌發(fā)生EMT[55]。

miRNA還可通過直接調控相關轉錄因子進而作用于 CDH1。據(jù)報道,幾種誘發(fā) EMT的因子,如ZEB1、ZEB2、Twist1、Snai1、Snai2、TGF-β 和 E47均可通過與 CDH1啟動子區(qū)的 E-box結合抑制CDH1轉錄,促進腫瘤侵襲和轉移[56～58]。

在EMT中,miR-200家族是研究最多的miRNA[59]。miR-200家族包括 miR-200a、-200b、-200c、-141和-429。miR-200家族直接結合ZEB1和ZEB2 mRNA 3′UTR,進而抑制其表達[48],間接上調 CDH1表達,最終抑制EMT過程。同時,ZEB1和ZEB2反過來又可結合miR-200家族成員啟動子中的E-box,從而抑制miR-200家族成員的表達,這表明 miR-200家族和ZEB因子相互負調控,它們之間形成了一個負反饋環(huán)路[42]。Gibbons等[60]對 40株人非小細胞肺癌(Non-small cell lung cancer,NSCLC)細胞進行了分析,發(fā)現(xiàn)miR-200家族的表達和EMT標志物具有相關性。此后,他們又將小鼠K-RAS和p53基因突變獲得肺腺癌模型后,用 TGF-β處理小鼠誘導其發(fā)生EMT,使得 miR-200家族低表達,而上調 miR-200家族的表達會阻礙EMT的發(fā)生,這表明NSCLC發(fā)生 EMT這一過程取決于 miR-200家族的表達。在miR-200家族上游調控方面,Yang等[61]發(fā)現(xiàn)具有轉移傾向的鼠肺腺癌細胞能高表達Notch和Notch配體,其中Notch配體Jagged2能上調GATA結合因子的表達,而GATA結合因子可以抑制EMT轉錄抑制因子 miR-200家族的表達,從而誘導 EMT,同時miR-200又可調節(jié)Gata3的表達,他們互相調控。這些結果表明,miR-200家族有可能成為肺癌治療的潛在靶點。

最近,Kumarswamy等[39]通過生物信息學方法分析發(fā)現(xiàn),在NSCLC細胞株中,Snail的上游調控基因 miR-30a與 CDH1和 N-鈣粘蛋白的表達相關,miR-30a可靶向CDH1的轉錄抑制因子Snai1來抑制EMT,而且與相應的正常組織比較,miR-30a在NSCLC中表達下調。在非肺癌組織和細胞中,有研究證實miR-30a可以靶向Snai1阻斷TGF-β誘導的EMT過程[62]。肺癌發(fā)生 EMT,除了 miR-30a,miR-34[50]也可作用于CDH1轉錄抑制物Snai1。miR-10b是Twist1的直接靶點,但是單獨改變miR-10b的表達并不能誘導人類乳腺癌細胞發(fā)生EMT[35]。這表明,miRNA在 EMT過程中的調控作用并非唯一,有其他基因的參與,甚至是一個網絡。

3.2 miRNA直接靶向DICER調節(jié)EMT

Dicer是一種核糖核酸內切酶,屬于 RNase III家族中特異識別雙鏈 RNA的一員。Bernstein等[63]首次在小干擾RNA(Small interfering RNA,siRNA)形成過程中明確了其作用,在胞質中其可對 premiRNA進行剪切形成成熟的miRNA[64,65]。在腫瘤的基因組中經常會出現(xiàn) Dicer1的缺失突變,上調Dicer1的表達則會抑制腫瘤的形成[66],反之亦然[67]。在間質型或發(fā)生遠處骨轉移的乳腺癌細胞中,Dicer1呈現(xiàn)低表達[68]。這些研究表明,Dicer1在腫瘤中具有雙面作用：它是腫瘤細胞增殖和細胞存活所不可或缺的,同時又是腫瘤向遠處轉移的一道屏障。

Dicer1是 miR-103/107的靶基因,其表達高低和腫瘤轉移及乳腺癌患者的預后緊密相關。在穩(wěn)定表達 miR-103/107的MDA-MB-231細胞中,Dicer1和成熟 miRNA的表達均下降。在臨床上,高表達miR-103/107的乳腺癌患者發(fā)生轉移的機率更高。同時,miR-103/107高表達會誘發(fā) EMT,這是由于miR-107可通過miR-200通路調節(jié)ZEB1/ZEB2的表達[51]?？傊?miR-103/miR-107可以調節(jié)Dicer1的表達,進而減少成熟miRNA的生物合成,最終增強細胞的遷移能力及體內腫瘤的轉移。

Su等[69]研究表明,TAp63可調控Dicer1的表達,Dicer低表達會增加細胞的侵襲能力,同時TAp63可以反式激活miR-130b的表達,miR-130b與轉移密切相關并且其加工成熟需Dicer1的參與。在TAp63缺失表達的細胞中上調 Dicer1和 miR-130b的表達顯著影響其轉移能力。通過調控 Dicer1和 miR-130b的表達,TAp63可抑制腫瘤的形成和轉移。

miR-18a作為癌基因miR-17-92簇的一員,在鼻咽癌組織及細胞中呈現(xiàn)高表達,并且與中晚期鼻咽癌發(fā)展密切相關。研究表明,miR-18a可與Dicer1基因 3′UTR結合抑制 Dicer1翻譯,因而包括 miR-200家族和miR-143在內的miRNA合成受到影響,最終誘發(fā)EMT,促進腫瘤轉移[52]。因此,miRNA可通過調控Dicer1的表達影響EMT。

3.3 miRNA通過鋅指蛋白調節(jié)EMT

鋅指蛋白(Tristetraprolin,TTP)是一種可降解靶基因mRNA 3′UTR中富含AU序列的蛋白。在Ras信號通路的作用下,下調TTP可以引起EMT和癌癥轉移。miR-29a通過結合3′UTR的2個位點調節(jié)TTP的表達,因而miR-29a可通過TTP參與EMT及乳腺癌的轉移[53]。

3.4 miRNA通過NF-κB調節(jié)EMT

NF-κB 是一個蛋白復合體,在 DNA 轉錄成mRNA過程中起調控作用。在多種腫瘤中,NF-κB出現(xiàn)異常(包括表達改變和位置變化),并且和轉移有關[70～73]。在化療反應中,NF-κB和miR-448之間可形成正反饋環(huán)路,通過EMT調節(jié)腫瘤轉移[54]。

4 miRNA參與腫瘤轉移其他步驟的調控

腫瘤轉移是一個多步驟的過程,包括腫瘤細胞從原發(fā)部位擴散,入侵組織,滲入血管或淋巴管,隨血液循環(huán)系統(tǒng)或淋巴循環(huán)系統(tǒng)在體內播散,遷出血管或淋巴管以及在新的地點定居和增殖[2]。除EMT外,miRNA也參與腫瘤轉移所必須經歷的其他過程(表2),如細胞外基質降解和血管生成等方面。眾所周知,腫瘤細胞在轉移過程中需要將細胞外基質蛋白降解才可出入自由,參與這一過程的miRNA包括 miR-335[74]、miR-373和 miR-520c[75]。腫瘤在發(fā)生微轉移后,血管生成是其生長存活所必須的,miR-9在乳腺癌中高表達,其在轉移中具有雙重作用,不但引發(fā)細胞 EMT,而且刺激血管的形成[46]。miR-9介導的CDH1下降會激活β-catenin信號通路,使血管內皮生長因子表達上升,最終導致血管生成增加。有研究表明,miR-126[76,77]和miR-205[78]也調節(jié)血管生成。因此,miRNA可調控ECM降解和血管形成繼而影響腫瘤轉移。

表2 在腫瘤轉移中起作用的其他 miRNA(不包括調控EMT/MET的miRNA)

5 結 語

miRNAs可以作為潛在的癌基因或者抑制基因在EMT過程中起作用,可作為腫瘤惡化或者逆轉的生物標志物。今后的研究無疑是要將注意力集中在不同腫瘤 EMT中發(fā)生特異性改變的 miRNA,以及這些 miRNA下游和上游調控相關的一些尚未被發(fā)現(xiàn)的新的調控因子上。EMT是一個多步驟過程,而這個過程涉及到許多信號分子、通路和轉錄因子。更為重要的是,EMT也和轉移、耐藥以及腫瘤干細胞的形成密切相關。越來越多的證據(jù)表明,促進EMT和腫瘤干細胞形成的治療性藥物[83]以及分子靶向治療也可促進轉移[84]。鑒于EMT在腫瘤發(fā)展中的重要作用,針對參與EMT的蛋白和miRNA可以提供一個具有特異性的治療策略,以防止腫瘤的轉移、耐藥和復發(fā)[85]。這些新的治療策略將開辟癌癥個性化治療的新方向,將提高人們對腫瘤EMT和轉移的認識,最終在腫瘤綜合診治的方案及策略選擇上有所突破。

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