杨丽华,沈星凯,李静秋,杨杰,乐燕萍,龚朝辉

1. 宁波大学医学院生物化学与分子生物学研究所,宁波 315211;

2. 浙江省病理生理学技术研究重点实验室,宁波 315211

据估计,2013年仅美国就有580350人死于癌症,平均每天约1600人,癌症已成为仅次于心脏疾病的第二大死因[1]。在我国,根据中国卫生统计年鉴(2012 版)统计,1973～1975、1990～1992、2004～2005年中国恶性肿瘤死亡抽样回顾调查结果显示,恶性肿瘤死亡率呈显著上升趋势,分别为 83.65/100000、108.26/100000和134.80/100000。癌症致人死亡的原因近 90%是癌症发生了转移。转移是恶性肿瘤最显著的特征之一,它是一个多步骤的过程[2]。肿瘤细胞发生转移的初始会发生上皮间质转化(Epithelialmesenchymal transition,EMT),EMT过程又受到多种因素调控,如转录因子、微RNA(MicroRNA,miRNA)等。miRNA是一类内源性非编码小分子RNA,与细胞生长、凋亡和信号转导密切相关。同时,miRNA可以调控EMT过程,在肿瘤转移发面发挥了重要作用。

1 EMT和肿瘤转移

EMT是胚胎正常发育、伤口愈合和恶性上皮肿瘤发生过程中的一个极其重要的基本过程[3],它可以使上皮细胞发生形态改变,转换成有运动能力的间充质细胞[4]。这些改变会使细胞-细胞之间和细胞-细胞外基质之间的连接受到破坏,因而细胞可以迁移到身体的其他部位参与组织重建和修复[5]。EMT在肿瘤细胞的迁移/侵袭以及多种癌症进程方面也起到非常重要的作用,是肿瘤细胞转移中的一个关键步骤,也是一个特定的信号通路[6,7]。EMT的特点是细胞粘附能力丧失,E-钙粘蛋白(E-cadherin,CDH1)表达下降,间叶标志物(包括 N-钙粘蛋白(N-cadherin)、波形蛋白(Vimentin)和纤维连接蛋白(Fibronectin)表达上升,细胞运动能力和侵袭能力增强[8]。原发部位的肿瘤细胞可以发生EMT来获得运动能力和侵袭能力,而间质上皮转化(Mesenchymalepithelial transition,MET)在肿瘤转移的最后一步起到重要作用,即从血管或淋巴管中渗出的肿瘤细胞会恢复到上皮细胞状态,然后增殖形成巨大的甚至肉眼可见的继发性肿瘤[9]。肿瘤EMT过程受内在因素和外在因素或者是二者共同作用的影响(图1),其中内在因素包括肿瘤发生过程中产生的基因突变。突变的K-RAS途径即是一个典型的例子[10],它可以单独诱导 EMT,也可以联合 EMT诱导因子如转化生长因子-β(Transforming growth factor-β,TGF-β)或者缺氧诱导因子 -2α(Hypoxia-inducible factor-2α,HIF-2α)共同作用。在某种程度上,由烟草(Nicotiana tabacum L.)引起的缺氧或者炎性肿瘤微环境,通过细胞外信号通路的失调,创造一个促进EMT发生的环境,特别是 TGF-β和环氧化酶/前列腺素 E2途径在肿瘤微环境中具有重要作用[11]。在肺癌中,缺氧肿瘤微环境诱导EMT主要是上调了碱性螺旋-环-螺旋类(Basic helix-loop helix,bHLH)转录因子和HIF的表达。在EMT的细胞内外信号通路中,EMT诱导转录因子和 miRNA起主要作用。在多发性肿瘤中,EMT被认为是转移的标志,与许多转录因子紧密相关。已有研究表明,转录因子能够促进 EMT和肿瘤转移[12～16]。转移过程中的多个步骤都受到这些转录因子调节,而这些因子本身的表达同时受到miRNA的精细调控。

图1 癌变过程中多种因素共同诱导EMT

2 影响EMT过程的转录因子

影响 EMT的转录抑制因子包括 Snail1[12]、Snail2[13]、Twist1[14]、Zeb1[15]、Zeb2[16]、Goosecoid[17]、FOXC2[18]、FoxQ1[19]、KLF8[20]、Prrx1[21]等。本文主要总结了 Snail、Twist和 Zeb转录因子家族及其在EMT和肿瘤转移方面的网络调控作用。

2.1 Snail锌指转录抑制因子家族

锌指簇可以与靶基因调控区的 E-box结合进行调控,Snail家族是EMT转录抑制因子中研究最多的。诸多研究表明,很多信号通路会诱导 Snail1和Snail2 的表达,包括 TGF-β[22]、Notch[23]和 Wnt[24]信号通路,活性氧[25]和缺氧[26]。其中,TGF-β信号通路是最重要的EMT诱导细胞因子之一,它可以激活包括Snail1/2在内的一系列EMT转录因子[27,28]。肝纤维化模型在缺氧条件下,基质细胞会分泌大量的TGF-β,随后肿瘤细胞受到 TGF-β的刺激后,促进Snail1表达,诱发EMT[29],这表明EMT诱导的各种信号通路之间互有关联。

2.2 Twist bHLH转录因子家族

参与EMT调控的bHLH转录因子包括E12和E47(E2A基因选择性剪切产物)、Twist1和2、Id1-4[30],均可以直接负调控CDH1的表达。Twist1和Twist2表达异常诱发 EMT的发生[14,31]。这是由于 Twist1可以结合Snail2启动子区,从而促进Snail2基因转录,过表达的 Snail2蛋白介导乳腺上皮肿瘤细胞发生EMT[32]。同时,Twist1亦可通过直接与血小板衍生生长因子受体α(Platelet-derived growth factor receptor-alpha,PDGFRα)启动子区结合,促进 PDGFα及其下游基因的表达,促进侵袭性伪足的形成,引起肿瘤细胞的侵袭和转移[33]。在干细胞和癌症中经常呈现高表达的多梳抑制复合体蛋白 Bmi1同Twist1形成复合体,进而抑制CDH1和p16INK4a基因表达[34]。Twist亦可诱导miR-10b的表达,miR- 10b通过靶向抑制Homebox D10的翻译,造成促转移基因RHOC的表达上调,最终影响乳腺癌转移[35]。一些癌基因可以诱导细胞异常增殖和异常转化,但细胞衰老会抑制肿瘤发生EMT和转移[36]。Twist2亦可下调CDH1的表达,参与TGF-β和p21诱导结肠癌细胞发生EMT这一过程[37]。在转录水平,Twist1主要受缺氧信号通路的调控[38]。在转录后水平,几种微RNA可以直接靶向Twist1 mRNA[39]。此外,通过激酶作用,Twist1蛋白在 Ser68处发生磷酸化,使Twist1更稳定,并且促进乳腺癌细胞的侵袭[40]。以上研究表明,Twist bHLH家族通过EMT影响肿瘤转移。

2.3 Zeb锌指转录因子家族

Zeb家族包括锌指/同源结构域蛋白-ZEB1和ZEB2,在物种间高度保守,和其他转录因子共同作用调节EMT,并且在这一过程中受SUMO化、乙酰化和磷酸化的调控。这些蛋白均可通过抑制上皮型标志物和激活间质型标志物的表达来诱发 EMT过程。多种因子和信号通路可直接激活Zeb家族的表达,如生长激素和类固醇激素、缺氧条件下的HIF-1α、炎性细胞因子、配体(成纤维细胞生长因子、胰岛素样生长因子-1、血小板衍生生长因子等受体的配体)、Ras-ERK2-Fra1、NF-κB 和 JAK/STAT3 等肿瘤中经常处于激活状态的下游通路、Wnt和Notch信号通路。Zeb家族也受miR-200的抑制,它们之间存在一个负反馈环路,最终诱发多种肿瘤的EMT[41,42]。

3 miRNA通过EMT调节肿瘤转移

miRNA是一类长约 22 nt的小分子RNA,可在转录后水平通过结合靶 mRNA的 3′UTR阻止靶mRNA的翻译或者促进靶mRNA的降解,实现对其靶基因的负调控[43]。据估计,miRNA可以调控人类30%左右蛋白的表达[44]。miRNA通过调节肿瘤细胞EMT相关靶蛋白表达,进而抑制或促进肿瘤转移(表 1)。

3.1 miRNA通过 CDH1、波形蛋白和转录因子调节EMT

CDH1是 EMT/MET中非常重要的调控基因,miRNA可以通过多种机制对其进行调节。miR-9通过直接靶向CDH1 mRNA引发EMT,促进乳腺癌转移。和未发生转移的乳腺癌患者相比,发生转移患者的原位癌组织中miR-9的表达升高。进一步研究发现,miR-9增加细胞的运动和侵袭能力,因而使非转移的乳腺癌细胞形成微转移[46]。miR-23a也直接靶向CDH1调控肺癌发生EMT[49]。Xu等[45]研究表明,miR-25在食管鳞状细胞癌中表达上调,其表达和淋巴结转移及肿瘤分期相关,这是由于miR-25直接负调控CDH1,进而促进细胞侵袭和迁移。

表1 参与肿瘤EMT的miRNA

miRNA除了直接靶向CDH1外,亦可间接调控CDH1。miR-138表达下调会促进 EMT的发生,其直接靶点是波形蛋白[47]。除了波形蛋白,miR-138还靶向EMT相关的其他靶基因,如ZEB2和靶向zeste基因增强子人类同源物2 (Enhancer of zeste homologue 2,EZH2)。由于EZH2可抑制组蛋白去乙酰化酶1/2(Histone deacetylases 1/2,HDAC1/2)和Snail的表达,间接降低 CDH1的表达,最终诱发鼻咽癌发生EMT[55]。

miRNA还可通过直接调控相关转录因子进而作用于 CDH1。据报道,几种诱发 EMT的因子,如ZEB1、ZEB2、Twist1、Snai1、Snai2、TGF-β 和 E47均可通过与 CDH1启动子区的 E-box结合抑制CDH1转录,促进肿瘤侵袭和转移[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家族直接结合ZEB1和ZEB2 mRNA 3′UTR,进而抑制其表达[48],间接上调 CDH1表达,最终抑制EMT过程。同时,ZEB1和ZEB2反过来又可结合miR-200家族成员启动子中的E-box,从而抑制miR-200家族成员的表达,这表明 miR-200家族和ZEB因子相互负调控,它们之间形成了一个负反馈环路[42]。Gibbons等[60]对 40株人非小细胞肺癌(Non-small cell lung cancer,NSCLC)细胞进行了分析,发现miR-200家族的表达和EMT标志物具有相关性。此后,他们又将小鼠K-RAS和p53基因突变获得肺腺癌模型后,用 TGF-β处理小鼠诱导其发生EMT,使得 miR-200家族低表达,而上调 miR-200家族的表达会阻碍EMT的发生,这表明NSCLC发生 EMT这一过程取决于 miR-200家族的表达。在miR-200家族上游调控方面,Yang等[61]发现具有转移倾向的鼠肺腺癌细胞能高表达Notch和Notch配体,其中Notch配体Jagged2能上调GATA结合因子的表达,而GATA结合因子可以抑制EMT转录抑制因子 miR-200家族的表达,从而诱导 EMT,同时miR-200又可调节Gata3的表达,他们互相调控。这些结果表明,miR-200家族有可能成为肺癌治疗的潜在靶点。

最近,Kumarswamy等[39]通过生物信息学方法分析发现,在NSCLC细胞株中,Snail的上游调控基因 miR-30a与 CDH1和 N-钙粘蛋白的表达相关,miR-30a可靶向CDH1的转录抑制因子Snai1来抑制EMT,而且与相应的正常组织比较,miR-30a在NSCLC中表达下调。在非肺癌组织和细胞中,有研究证实miR-30a可以靶向Snai1阻断TGF-β诱导的EMT过程[62]。肺癌发生 EMT,除了 miR-30a,miR-34[50]也可作用于CDH1转录抑制物Snai1。miR-10b是Twist1的直接靶点,但是单独改变miR-10b的表达并不能诱导人类乳腺癌细胞发生EMT[35]。这表明,miRNA在 EMT过程中的调控作用并非唯一,有其他基因的参与,甚至是一个网络。

3.2 miRNA直接靶向DICER调节EMT

Dicer是一种核糖核酸内切酶,属于 RNase III家族中特异识别双链 RNA的一员。Bernstein等[63]首次在小干扰RNA(Small interfering RNA,siRNA)形成过程中明确了其作用,在胞质中其可对 premiRNA进行剪切形成成熟的miRNA[64,65]。在肿瘤的基因组中经常会出现 Dicer1的缺失突变,上调Dicer1的表达则会抑制肿瘤的形成[66],反之亦然[67]。在间质型或发生远处骨转移的乳腺癌细胞中,Dicer1呈现低表达[68]。这些研究表明,Dicer1在肿瘤中具有双面作用：它是肿瘤细胞增殖和细胞存活所不可或缺的,同时又是肿瘤向远处转移的一道屏障。

Dicer1是 miR-103/107的靶基因,其表达高低和肿瘤转移及乳腺癌患者的预后紧密相关。在稳定表达 miR-103/107的MDA-MB-231细胞中,Dicer1和成熟 miRNA的表达均下降。在临床上,高表达miR-103/107的乳腺癌患者发生转移的机率更高。同时,miR-103/107高表达会诱发 EMT,这是由于miR-107可通过miR-200通路调节ZEB1/ZEB2的表达[51]。总之,miR-103/miR-107可以调节Dicer1的表达,进而减少成熟miRNA的生物合成,最终增强细胞的迁移能力及体内肿瘤的转移。

Su等[69]研究表明,TAp63可调控Dicer1的表达,Dicer低表达会增加细胞的侵袭能力,同时TAp63可以反式激活miR-130b的表达,miR-130b与转移密切相关并且其加工成熟需Dicer1的参与。在TAp63缺失表达的细胞中上调 Dicer1和 miR-130b的表达显著影响其转移能力。通过调控 Dicer1和 miR-130b的表达,TAp63可抑制肿瘤的形成和转移。

miR-18a作为癌基因miR-17-92簇的一员,在鼻咽癌组织及细胞中呈现高表达,并且与中晚期鼻咽癌发展密切相关。研究表明,miR-18a可与Dicer1基因 3′UTR结合抑制 Dicer1翻译,因而包括 miR-200家族和miR-143在内的miRNA合成受到影响,最终诱发EMT,促进肿瘤转移[52]。因此,miRNA可通过调控Dicer1的表达影响EMT。

3.3 miRNA通过锌指蛋白调节EMT

锌指蛋白(Tristetraprolin,TTP)是一种可降解靶基因mRNA 3′UTR中富含AU序列的蛋白。在Ras信号通路的作用下,下调TTP可以引起EMT和癌症转移。miR-29a通过结合3′UTR的2个位点调节TTP的表达,因而miR-29a可通过TTP参与EMT及乳腺癌的转移[53]。

3.4 miRNA通过NF-κB调节EMT

NF-κB 是一个蛋白复合体,在 DNA 转录成mRNA过程中起调控作用。在多种肿瘤中,NF-κB出现异常(包括表达改变和位置变化),并且和转移有关[70～73]。在化疗反应中,NF-κB和miR-448之间可形成正反馈环路,通过EMT调节肿瘤转移[54]。

4 miRNA参与肿瘤转移其他步骤的调控

肿瘤转移是一个多步骤的过程,包括肿瘤细胞从原发部位扩散,入侵组织,渗入血管或淋巴管,随血液循环系统或淋巴循环系统在体内播散,迁出血管或淋巴管以及在新的地点定居和增殖[2]。除EMT外,miRNA也参与肿瘤转移所必须经历的其他过程(表2),如细胞外基质降解和血管生成等方面。众所周知,肿瘤细胞在转移过程中需要将细胞外基质蛋白降解才可出入自由,参与这一过程的miRNA包括 miR-335[74]、miR-373和 miR-520c[75]。肿瘤在发生微转移后,血管生成是其生长存活所必须的,miR-9在乳腺癌中高表达,其在转移中具有双重作用,不但引发细胞 EMT,而且刺激血管的形成[46]。miR-9介导的CDH1下降会激活β-catenin信号通路,使血管内皮生长因子表达上升,最终导致血管生成增加。有研究表明,miR-126[76,77]和miR-205[78]也调节血管生成。因此,miRNA可调控ECM降解和血管形成继而影响肿瘤转移。

表2 在肿瘤转移中起作用的其他 miRNA(不包括调控EMT/MET的miRNA)

5 结 语

miRNAs可以作为潜在的癌基因或者抑制基因在EMT过程中起作用,可作为肿瘤恶化或者逆转的生物标志物。今后的研究无疑是要将注意力集中在不同肿瘤 EMT中发生特异性改变的 miRNA,以及这些 miRNA下游和上游调控相关的一些尚未被发现的新的调控因子上。EMT是一个多步骤过程,而这个过程涉及到许多信号分子、通路和转录因子。更为重要的是,EMT也和转移、耐药以及肿瘤干细胞的形成密切相关。越来越多的证据表明,促进EMT和肿瘤干细胞形成的治疗性药物[83]以及分子靶向治疗也可促进转移[84]。鉴于EMT在肿瘤发展中的重要作用,针对参与EMT的蛋白和miRNA可以提供一个具有特异性的治疗策略,以防止肿瘤的转移、耐药和复发[85]。这些新的治疗策略将开辟癌症个性化治疗的新方向,将提高人们对肿瘤EMT和转移的认识,最终在肿瘤综合诊治的方案及策略选择上有所突破。

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