microRNA在腫瘤中的作用*

張冰潔金由辛馬中良

①碩士研究生，②教授，③副教授，上海大學(xué)生命科學(xué)學(xué)院，上海 200444

*上海市科委自然科學(xué)基金(11ZR141220)和國(guó)家自然科學(xué)基金(31170750)資助

microRNA在腫瘤中的作用*

張冰潔①金由辛②馬中良③

①碩士研究生，②教授，③副教授，上海大學(xué)生命科學(xué)學(xué)院，上海 200444

*上海市科委自然科學(xué)基金(11ZR141220)和國(guó)家自然科學(xué)基金(31170750)資助

miRNA；腫瘤；預(yù)測(cè)；診斷；治療；預(yù)后

miRNA(microRNAs)是一類由內(nèi)源基因編碼的長(zhǎng)度為18～23個(gè)堿基的非編碼單鏈RNA分子，它們?cè)谵D(zhuǎn)錄后水平調(diào)節(jié)基因的表達(dá)。據(jù)報(bào)道，在多種癌癥中發(fā)現(xiàn)了miRNA表達(dá)量改變，提示其可能與癌癥的發(fā)病機(jī)理、腫瘤生長(zhǎng)和轉(zhuǎn)移等相關(guān)，起到癌基因或抑癌基因的作用。大量證據(jù)表明，miRNA的異常表達(dá)會(huì)促進(jìn)腫瘤的發(fā)生和發(fā)展，因此在包括非小細(xì)胞肺癌、乳腺癌和前列腺癌等多種癌癥中具有臨床價(jià)值。筆者主要闡述miRNA的生成過(guò)程，以及目前已知的相關(guān)miRNA在肺癌、乳腺癌、前列腺癌的預(yù)測(cè)、診斷、治療和預(yù)后中的臨床應(yīng)用及相關(guān)分子機(jī)制。

癌癥是嚴(yán)重影響人類健康的重大疾病，其中肺癌、乳腺癌、前列腺癌等均為常見的惡性腫瘤。miRNA的發(fā)現(xiàn)為癌癥的治療開發(fā)了新的途徑。miRNA幾乎參與了所有的基礎(chǔ)信號(hào)通路，包括調(diào)控與腫瘤發(fā)生有關(guān)的許多重要基因的表達(dá)[1]。筆者將探討目前已知的相關(guān)miRNA在肺癌、乳腺癌、前列腺癌等的預(yù)測(cè)、診斷、治療和預(yù)后中的臨床應(yīng)用及相關(guān)分子機(jī)制。

1 miRNA的特性、發(fā)現(xiàn)與命名

miRNA是一類由內(nèi)源基因編碼的長(zhǎng)度為18～23個(gè)堿基的非編碼單鏈RNA分子，它們參與生物體內(nèi)的多種進(jìn)程，如器官形態(tài)形成與改變、免疫系統(tǒng)發(fā)育、造血、新陳代謝、應(yīng)激反應(yīng)、過(guò)渡轉(zhuǎn)變、雙邊不對(duì)稱、細(xì)胞分化、增殖和凋亡等。miRNA能在轉(zhuǎn)錄后水平調(diào)節(jié)基因表達(dá)，從而對(duì)細(xì)胞進(jìn)行基礎(chǔ)性調(diào)控。

miRNA具有高度保守性、序列同源性、時(shí)序性及組織特異性，這些性質(zhì)與其功能密切相關(guān)。生物信息學(xué)數(shù)據(jù)顯示，miRNA至少能夠調(diào)節(jié)20%～30%的人類基因。單個(gè)miRNA能調(diào)節(jié)多達(dá)100個(gè)不同的mRNA，并且數(shù)據(jù)顯示有1 000個(gè)以上的mRNA受同一個(gè)miRNA的調(diào)控。miRNA參與多種細(xì)胞進(jìn)程的調(diào)控，如細(xì)胞增殖、凋亡、細(xì)胞周期和分化等。miRNA異常表達(dá)調(diào)控與腫瘤、心腦血管疾病等多種重大疾病的發(fā)生有關(guān)。miRNA既可作為抑癌基因下調(diào)原癌基因活性，也可作為癌基因下調(diào)抑癌基因活性，還可調(diào)節(jié)腫瘤相關(guān)基因的表達(dá)，其自身突變、缺失、易位及相互調(diào)控異常等還可導(dǎo)致相關(guān)基因異常表達(dá)。大量研究表明[2-4]，miRNA在人類多種腫瘤中表達(dá)的改變，與腫瘤發(fā)生、發(fā)展、診斷、治療及預(yù)后密切相關(guān)。

Victor Ambros 實(shí)驗(yàn)室于 1993 年在秀麗隱桿線蟲 (Caenorbabditis elegans) 中首次發(fā)現(xiàn)了miRNA，并將之命名為lin-4，它是線蟲發(fā)育時(shí)序的一個(gè)調(diào)控因子[5]。與此同時(shí)，Gary Ruvkun的實(shí)驗(yàn)室鑒定了首個(gè)miRNA的靶標(biāo)基因[6]。這兩個(gè)重要的發(fā)現(xiàn)共同確認(rèn)了一種新的轉(zhuǎn)錄后基因調(diào)控機(jī)制。然而，當(dāng)時(shí)科學(xué)家們并未察覺(jué)到miRNA的重要性。時(shí)隔7年，當(dāng)Reinhart及其同事在線蟲中鑒定出第2個(gè)miRNA——let-7[7]，以及l(fā)et-7與當(dāng)時(shí)已引起人們興趣的另一類小RNA——siRNA的關(guān)系時(shí)，科學(xué)家們才意識(shí)到miRNA的重要性，由此開啟了一個(gè)全新的轉(zhuǎn)錄后基因調(diào)控時(shí)代。隨后，在包括從線蟲、果蠅到人類范圍的多個(gè)物種中都發(fā)現(xiàn)了miRNA[8]，表明這些分子代表一個(gè)基因家族，由一個(gè)古老的祖先小分子RNA基因進(jìn)化而來(lái)。

當(dāng)實(shí)驗(yàn)人員開始對(duì)數(shù)目日益增多的miRNA進(jìn)行克隆和測(cè)序時(shí)，建立一個(gè)對(duì)已公布miRNA進(jìn)行命名、注釋和分類的專業(yè)數(shù)據(jù)庫(kù)變得日益迫切，于是miRBase數(shù)據(jù)庫(kù)應(yīng)運(yùn)而生。miRBase數(shù)據(jù)庫(kù)是miRNA序列和注釋的主要在線儲(chǔ)存庫(kù)，供人們查詢已收錄miRNA的序列、基因組定位、前體的基因組織形式等相關(guān)信息。要建立數(shù)據(jù)庫(kù)首先需對(duì)miRNA進(jìn)行系統(tǒng)命名。miRNA成熟體的命名格式為：XXXX-miR-YYYY?！癤XXX”代表由3到4個(gè)字母組成的物種代碼，“YYYY”是順序的數(shù)字識(shí)別號(hào)，例如hsa-miR-21。對(duì)于高度同源的miRNA，則在其數(shù)字識(shí)別號(hào)后加上英文小寫字母加以區(qū)分，如hsa-miR-34a、hsa-miR-34b等。由不同轉(zhuǎn)錄本加工而成的具有相同成熟序列的miRNA，則在其后加上阿拉伯?dāng)?shù)字以示區(qū)別，如hsa-miR-519a-1、hsa-miR-519a-2。由于miRNA命名原則確定之前，lin-4和let-7已被廣泛接受，因此保留原名。

2 miRNA的生成與作用機(jī)制

在哺乳動(dòng)物中，miRNA的合成分別在細(xì)胞質(zhì)和細(xì)胞核中進(jìn)行(圖1)[9]。在細(xì)胞核中，編碼miRNA的基因首先在RNA聚合酶II的作用下，產(chǎn)生初始miRNA(pri-miRNA)。然后，在Drosha核酸酶及DGCB8組成的復(fù)合體作用下，pri-miRNA被剪切成長(zhǎng)度為70～100個(gè)核苷酸、具莖環(huán)結(jié)構(gòu)的前體miRNA(pre-miRNA)。pre-miRNA被Ran-GTP依賴的轉(zhuǎn)運(yùn)蛋白Exportin-5特異性識(shí)別后從細(xì)胞核運(yùn)送到細(xì)胞質(zhì)內(nèi)。隨后，pre-miRNA在胞質(zhì)中被Dicer核酸酶切除末端莖環(huán)結(jié)構(gòu)，并在雙鏈RNA結(jié)合蛋白Loquacious的輔助下，釋放出約22 bp miRNA: miRNA*雙體。之后，成熟的miRNA進(jìn)入RNA誘導(dǎo)的基因沉默復(fù)合物(RNA-induced silencing complex, RISC)并參與形成非對(duì)稱RISC復(fù)合物(asymmetric RISC assembly)。成熟的miRNA單鏈與靶mRNA的3'-UTR上的同源序列部分或完全結(jié)合，阻礙翻譯的繼續(xù)或引起mRNA降解(少數(shù)情況下)，從而發(fā)揮廣泛的生物學(xué)作用。雙體中只有一條鏈能結(jié)合到RISC上形成成熟的miRNA，另一條鏈則被降解。兩條鏈形成成熟miRNA的機(jī)會(huì)是不均等的，這與它們的穩(wěn)定性不同密切相關(guān)。miRNA: miRNA*雙螺旋中兩條鏈并非完全配對(duì)，每條鏈的3'端均有2個(gè)游離的核苷酸，但miRNA鏈上靠近5'端有一個(gè)不與miRNA*配對(duì)的小突起，它明顯減弱了miRNA 5'端的穩(wěn)定性。由于成熟miRNA的產(chǎn)生總是偏向于選擇不穩(wěn)定的5'端，因此miRNA鏈被選中的概率遠(yuǎn)高于miRNA*鏈。

圖1 miRNA生成與作用機(jī)制

miRNA對(duì)靶基因調(diào)控主要通過(guò)與mRNA的3'-UTR互補(bǔ)配對(duì)來(lái)實(shí)現(xiàn)。若互補(bǔ)程度高，則降解靶mRNA；但在多數(shù)情況下，miRNA與靶mRNA為不完全互補(bǔ)配對(duì)，抑制其轉(zhuǎn)錄后的翻譯，而mRNA水平無(wú)顯著變化。2010年，Khraiwesh等[10]對(duì)miRNA的轉(zhuǎn)錄后調(diào)控機(jī)制做了更為深入的闡明。他們?cè)谛×⑼胩\(Physcomitrella patens)中研究發(fā)現(xiàn)，Dicer-like1基因編碼蛋白中，DICER-LIKE1a蛋白促進(jìn)miRNA成熟，而DICER-LIKE1b蛋白并不負(fù)責(zé)miRNA的成熟，控制miRNA對(duì)靶位的調(diào)控作用。一旦DICER-LIKE1b蛋白突變，將加劇miRNA:靶RNA雙鏈形成，導(dǎo)致編碼靶RNA的基因超甲基化，最終導(dǎo)致基因沉默，這些靶序列的轉(zhuǎn)錄率急劇降低。另有研究發(fā)現(xiàn)，miRNA與靶mRNA識(shí)別過(guò)程中，miRNA5'端的4～8位堿基在與靶mRNA的結(jié)合中比3'端序列更為重要，這段序列被稱為種子序列(seed sequence)，它在miRNA和靶mRNA配對(duì)中起十分關(guān)鍵的作用。由于大多數(shù)miRNA抑制基因的功能依賴于部分序列互補(bǔ)，單個(gè)miRNA能夠靶定多個(gè)mRNA，而多個(gè)miRNA也能同時(shí)作用于一個(gè)mRNA，從而在不同的組織和細(xì)胞中協(xié)同調(diào)節(jié)基因的表達(dá)強(qiáng)度[11]。所以，miRNA可能對(duì)蛋白質(zhì)編碼基因具有廣泛的微調(diào)作用。miRNA的發(fā)現(xiàn)改變了后基因組時(shí)代對(duì)基因調(diào)節(jié)的認(rèn)識(shí)和理解。

3 miRNA在腫瘤中的作用

全基因組研究顯示，miRNA基因通常定位于基因組中出現(xiàn)雜合缺失或擴(kuò)增的位點(diǎn)、脆性位點(diǎn)、病毒整合位點(diǎn)或其他癌癥相關(guān)的基因組區(qū)域，提示擾亂miRNA的表達(dá)和作用將促進(jìn)癌癥的發(fā)生與發(fā)展[12]。根據(jù)早期在腫瘤中的研究，通過(guò)對(duì)多種miRNA的表達(dá)進(jìn)行分析和研究發(fā)現(xiàn)，在多種實(shí)體瘤中miRNA的表達(dá)呈現(xiàn)大幅度改變現(xiàn)象。與傳統(tǒng)mRNA表達(dá)分析相比，miRNA表達(dá)譜能夠更好的分辨不同發(fā)育起源的腫瘤[13]。Volinia等[14]在540例腫瘤樣本(包括肺癌、乳腺癌、胃癌、前列腺癌、結(jié)腸癌和胰腺癌)中進(jìn)行大規(guī)模的miRNA表達(dá)分析，發(fā)現(xiàn)在實(shí)體瘤中大部分miRNA過(guò)表達(dá)這一特征。

腫瘤相關(guān)的miRNA被稱為腫瘤microRNA組學(xué)(onco-miR)。癌基因性質(zhì)的miRNA過(guò)表達(dá)會(huì)促進(jìn)腫瘤發(fā)生，它們通過(guò)作用于信號(hào)通路促進(jìn)細(xì)胞增殖、逃避細(xì)胞凋亡、激發(fā)無(wú)限復(fù)制潛能、促進(jìn)血管生成、侵襲和轉(zhuǎn)移等。同樣地，抑癌基因性質(zhì)的miRNA低表達(dá)也能產(chǎn)生相似的影響[15]。以miR-17～92家族為例，它們?yōu)榘┗蛐再|(zhì)的miRNA，在肺癌、B細(xì)胞淋巴瘤、乳腺癌和胰腺癌等幾種癌癥中，此家族的表達(dá)上調(diào)[14,16]。miR-17～92家族的靶基因包括E2F1、PTEN 和p21[17]。miR-92在肺癌，尤其是非小細(xì)胞肺癌(non-small-cell lung cancer，NSCLC)中高表達(dá)，它能在體外促進(jìn)細(xì)胞增殖[18]。在小鼠動(dòng)物實(shí)驗(yàn)中，miR-92表達(dá)缺失的轉(zhuǎn)基因組小鼠與正常小鼠相比表現(xiàn)出致死性，這些小鼠體型明顯偏小且肺臟嚴(yán)重發(fā)育不完全。除此之外，突變型小鼠的心臟在發(fā)育過(guò)程中也有缺陷[19]。Lu等[20]發(fā)現(xiàn)，miR-17～92在肺上皮組織中特定過(guò)表達(dá)導(dǎo)致胚胎致死性表型。其機(jī)制可能為miR-92通過(guò)調(diào)節(jié)Rb、Rbl1、Rbl2、PTEN基因從而起作用。

下面將分別介紹miRNA與肺癌、乳腺癌及前列腺癌的發(fā)病機(jī)理。

3.1 肺癌相關(guān)miRNA

目前，肺癌死亡率一直高居癌癥死亡率榜首[21]，患者5年生存率不到15%。約85%的肺癌為非小細(xì)胞肺癌，與小細(xì)胞癌相比其癌細(xì)胞生長(zhǎng)分裂較慢，擴(kuò)散轉(zhuǎn)移相對(duì)較晚。早期診斷發(fā)現(xiàn)可能發(fā)病的患者是NSCLC治療的最大挑戰(zhàn)之一。如果在原發(fā)位階段將其診出，則5年生存率將提升至近50%；若在發(fā)生淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移時(shí)才發(fā)現(xiàn)，那么其5年生存率將大大降低。尋找分子生物學(xué)標(biāo)記并將其應(yīng)用于診斷、治療和預(yù)后將極大地提高患者的生存率。

隨著基因組學(xué)和蛋白質(zhì)組學(xué)的發(fā)展，許多新的候選生物標(biāo)記物被發(fā)現(xiàn)，但仍然缺乏可靠的標(biāo)記物，對(duì)非小細(xì)胞肺癌的診斷依舊十分困難[13]。miRNA的發(fā)現(xiàn)與鑒定為肺癌的診斷提供了新的思路。

miR-21[22-29]、miR-34a[30-34]、miR-126[35-41]、miR-155[42-43]、miR-130[34]等是肺癌相關(guān)miRNA (表1)。miR-21在癌癥中一般為高表達(dá)。miR-21的表達(dá)上調(diào)通常與NSCLC病例的惡性結(jié)果相關(guān)[22-24]。miR-21可作為NSCLC復(fù)發(fā)與低生存率的預(yù)測(cè)因子[35-36]。miR-34a在肺癌中低表達(dá)，表明其在肺癌中起抑癌基因作用(筆者所在實(shí)驗(yàn)室的研究也證明了這一點(diǎn))。通過(guò)多變量分析發(fā)現(xiàn)，miR-34a有望成為NSCLC復(fù)發(fā)的獨(dú)立預(yù)測(cè)因子，并且可作為肺癌治療靶點(diǎn)。miR-126為抑癌基因，在肺癌中表達(dá)下調(diào)[35-41]。miR-126與NSCLC差預(yù)后性相關(guān)，證明其可作為NSCLC的特異性獨(dú)立預(yù)后因子。miR-155在NSCLC細(xì)胞中一般為高表達(dá)[42]，在NSCLC病例血清中低表達(dá)[43]。miR-155在臨床診斷及治療方面具有較高價(jià)值，深入研究其在肺癌中的作用機(jī)理、分子機(jī)制有助于明確其在肺癌中的作用及其在臨床方面的應(yīng)用。

表1 肺癌相關(guān)miRNA

3.2 乳腺癌相關(guān)miRNA

乳腺癌是女性最常見的惡性腫瘤，在女性中的死亡率僅次于肺癌[21,45]。2013年，美國(guó)新增乳腺癌病例達(dá)232 340例，占女性新發(fā)惡性腫瘤的29%，排名女性惡性腫瘤發(fā)病率首位[21]。在中國(guó)，乳腺癌的發(fā)病率和死亡率也居高不下，且發(fā)病率呈逐年升高趨勢(shì)。導(dǎo)致乳腺癌患者死亡的主要原因是癌癥轉(zhuǎn)移引起的并發(fā)癥[21]。乳腺癌的轉(zhuǎn)移是一個(gè)由多種基因調(diào)控、多步驟逐漸發(fā)展的復(fù)雜過(guò)程，涉及腫瘤細(xì)胞生長(zhǎng)、遷移和侵襲等一系列過(guò)程。miR-21[46-57]、miR-34a[58-62]、miR-126[63-66]、miR-155[67-69]、miR-200c[70-74]、miR-221/222[75-78]等多種miRNA參與了乳腺癌轉(zhuǎn)移的基因表達(dá)調(diào)控和信號(hào)通路調(diào)控等[63,70,79]，在腫瘤轉(zhuǎn)移過(guò)程中發(fā)揮重要作用(表2)。

miR-21在乳腺癌等多種癌癥中普遍高表達(dá)，已明確其為原癌基因作用的miRNA。miR-34a在多種癌癥中普遍低表達(dá)，是較為明確的抑癌基因。miR-34a表達(dá)激活是侵襲性乳腺癌表型的標(biāo)記之一，同時(shí)，還可作為乳腺癌低復(fù)發(fā)風(fēng)險(xiǎn)和低死亡率的獨(dú)立預(yù)測(cè)因子。miR-126在乳腺癌中低表達(dá)，且與乳腺癌的轉(zhuǎn)移密切相關(guān)。miR-126有可能是潛在的乳腺癌轉(zhuǎn)移生物標(biāo)記物。miR-155在乳腺癌中顯著高表達(dá)，是明確的原癌基因。Mattiske等[69]對(duì)miR-155進(jìn)行了深入全面的調(diào)查，總結(jié)了其在乳腺癌中的作用、分子機(jī)制和靶基因等，miR-155的靶基因有147個(gè)，如RhoA[67]、FOXO3A[68]、SOCS1[69]等。miR-200c在乳腺癌中低表達(dá)，提示其為抑癌基因作用的miRNA。miR-200c能夠抑制乳腺癌EMT進(jìn)程[70,80]。 miR-221/222在乳腺癌中普遍高表達(dá)。多項(xiàng)研究表明[75-76]，miR-221/222與乳腺癌的藥物抗性密切相關(guān)。

表2 乳腺癌相關(guān)miRNAs

圖2 部分相關(guān)miRNA在乳腺癌中的調(diào)控分子機(jī)理

3.3 前列腺癌相關(guān)miRNA前列腺癌是男性最常見的惡性腫瘤，是男性因癌癥死亡的第2大原因，僅次于肺癌[21,45]。2013年，美國(guó)新增前列腺癌病例238 590例，占男性新發(fā)惡性腫瘤的28%[21]。經(jīng)證實(shí)[81-83]，miRNA參與了前列腺癌發(fā)生、發(fā)展和轉(zhuǎn)移等一系列過(guò)程。miR-21[84-90]、miR-34a[81,91-94]、miR-221/222[95-97]、miR-205[46-48,95,98-100]等miRNA在前列腺癌中的作用詳見表3。

表3 前列腺癌相關(guān)miRNAs

4 展望

miRNA是一類非編碼單鏈RNA分子，它們通過(guò)調(diào)控基因表達(dá)和細(xì)胞內(nèi)信號(hào)通路參與腫瘤的發(fā)生和發(fā)展等一系列過(guò)程。miRNA的獨(dú)特生物學(xué)特性使其成為最為重要的腫瘤預(yù)測(cè)、診斷和預(yù)后的生物標(biāo)記物及治療靶點(diǎn)。然而，miRNA在腫瘤中的作用機(jī)制十分復(fù)雜，其所參與的調(diào)控網(wǎng)絡(luò)十分廣泛，同一miRNA在不同癌癥中的表達(dá)水平和作用可能不同，甚至同一miRNA在某種癌癥的不同分期/分型中的表達(dá)水平和作用也可能不同。深入透徹地研究特定miRNA在特定癌癥中的作用、分子機(jī)制和調(diào)控網(wǎng)絡(luò)有助于更好地了解癌癥的發(fā)病機(jī)理，并能更好地找出相應(yīng)的治療方法。

(2013年11月5日收稿)■

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The roles of microRNAs in cancer

ZHANG Bing-jie①, JIN You-xin②, MA Zhong-liang③
①M(fèi)aster Candidate, ②Professor, ③Associate Professor, School of Life Sciences, Shanghai University, Shanghai 200444, China

miRNAs, 18～23 nucleotide are a class of single stranded small non-coding RNA encoded by endogenous genes, which regulate gene expression at the post-transcriptional level. Abberant expressions of miRNAs reported in many kinds of human cancers may associate with cancer pathogenesis, tumor growth and metastasis, thus they can function as either oncogenes or antioncogenes. Numerous evidences indicated that the disregulation of miRNAs contribute to tumor initiation and progression. So these miRNAs can be used as clinical diagnosis biomarkers in many cancers, including non-small-cell lung cancer, breast cancer and prostate cancer. We introduce the clinical applications of the current known of miRNAs in early diagnosis, therapy and prognosis of non-small-cell lung cancer, breast cancer and prostate cancer, as well as their molecular mechanisms.

miRNA, cancer, predict, diagnosis, therapy, prognosis

(編輯：段艷芳)

10.3969/j.issn.0253-9608.2014.05.008