綜述審校

復(fù)旦大學(xué)附屬腫瘤醫(yī)院中西醫(yī)結(jié)合科，復(fù)旦大學(xué)上海醫(yī)學(xué)院腫瘤學(xué)系，上海 200032

外泌體源性非編碼RNA在消化系統(tǒng)惡性腫瘤中的研究進(jìn)展及展望

于淑林綜述，陳 震審校

復(fù)旦大學(xué)附屬腫瘤醫(yī)院中西醫(yī)結(jié)合科，復(fù)旦大學(xué)上海醫(yī)學(xué)院腫瘤學(xué)系，上海 200032

外泌體是納米級(jí)、雙層膜的囊泡，由多種細(xì)胞分泌，廣泛存在于多種體液中。外泌體中含有RNA和蛋白質(zhì)等多種活性物質(zhì)，在細(xì)胞物質(zhì)間和信號(hào)轉(zhuǎn)導(dǎo)中起重要作用。非編碼RNA具有重要的基因表達(dá)調(diào)控功能，在多種腫瘤的發(fā)生、發(fā)展過(guò)程中起重要作用。外泌體源性非編碼RNA的研究是目前新的研究熱點(diǎn)，現(xiàn)就外泌體及外泌體源性非編碼RNA在消化系統(tǒng)惡性腫瘤中的研究進(jìn)展進(jìn)行綜述。

外泌體；非編碼RNA；消化系統(tǒng)惡性腫瘤

外泌體是細(xì)胞經(jīng)過(guò)“內(nèi)吞－融合－外排”等一系列調(diào)控過(guò)程而形成的具有雙層膜結(jié)構(gòu)的囊泡，直徑約50～150 nm［1］，源于細(xì)胞內(nèi)吞系統(tǒng)的晚期內(nèi)體［2］。它富含蛋白、mRNA和核苷酸等多種成分［3］，能調(diào)節(jié)受體細(xì)胞的生物學(xué)行為［4］，發(fā)揮重要的調(diào)控作用。多種細(xì)胞可分泌外泌體，包括T細(xì)胞、血小板及腫瘤細(xì)胞等［5-9］。在人類基因組中，編碼蛋白質(zhì)的序列在基因組中所占的比例不到2%，其他超過(guò)90%的序列為非編碼區(qū)［10］。非編碼RNA作為一類特殊的RNA分子，包括微小RNA(microRNA，miRNA)、長(zhǎng)鏈非編碼RNA(long non-coding RNA， lncRNA)和環(huán)狀RNA(circle RNA，circRNA)在內(nèi)的多種RNA，具有調(diào)控基因表達(dá)、參與表觀遺傳修飾、細(xì)胞增殖及細(xì)胞凋亡等多種生命活動(dòng)的功能［11-12］。外泌體源性非編碼RNA在疾病中的研究是目前較新的研究領(lǐng)域，現(xiàn)就外泌體及外泌體源性非編碼RNA(包括miRNA、lncRNA和circRNA)在消化系統(tǒng)惡性腫瘤中的研究進(jìn)展進(jìn)行綜述。

1 外泌體與消化系統(tǒng)惡性腫瘤

消化系統(tǒng)惡性腫瘤，包括胃癌、肝癌、結(jié)直腸癌、胰腺癌和食管癌等，是危害人類健康的主要因素之一，約占腫瘤引起死亡人數(shù)的1/4，在我國(guó)發(fā)病率逐年上升，具有發(fā)病率高、癥狀隱匿及預(yù)后差等特點(diǎn)，其早期診斷、治療及預(yù)后是目前臨床消化系統(tǒng)腫瘤防治的重點(diǎn)［13］。包括外泌體在內(nèi)的液體活檢技術(shù)是目前的研究熱點(diǎn)，旨在尋找潛在非侵入性、創(chuàng)傷小、特異性高的診斷和篩查方法。

血液是唯一與所有器官都有接觸的組織，隨著檢測(cè)及分離技術(shù)日趨完善，外周血中的外泌體逐漸被人們了解。外周血中RNA的來(lái)源包括：細(xì)胞自身分泌或死亡破裂后所含的RNA直接釋放進(jìn)入循環(huán)，并可經(jīng)細(xì)胞死亡數(shù)量增加、細(xì)胞外RNA的含量也增加的研究證實(shí)；膜萌芽期RNA裝載于微囊泡中或裝載于外泌體中進(jìn)入外周血［14］。外泌體中的RNA雖然僅占外周血RNA的一小部分，但因其表面具有標(biāo)志性蛋白可進(jìn)行特異性富集，具有很好的腫瘤特異性。腫瘤細(xì)胞來(lái)源的外泌體既可以通過(guò)微環(huán)境介導(dǎo)腫瘤細(xì)胞的增生、血管形成及免疫抑制等腫瘤發(fā)生、發(fā)展的過(guò)程，又可以通過(guò)檢測(cè)其含有腫瘤細(xì)胞分泌的特異性成分(如非編碼RNA)，對(duì)腫瘤進(jìn)行診斷及監(jiān)測(cè)，為消化系統(tǒng)惡性腫瘤的診治提供較好的依據(jù)。

腫瘤細(xì)胞分泌的外泌體可以通過(guò)外泌體的自分泌信號(hào)通路來(lái)調(diào)節(jié)腫瘤的局部增生［15］。不同細(xì)胞類型及細(xì)胞特性來(lái)源的外泌體的自分泌作用存在差異。例如，源于胃癌細(xì)胞系的外泌體的自分泌信號(hào)可通過(guò)Akt和細(xì)胞外信號(hào)調(diào)節(jié)激酶磷酸化作用的增加來(lái)促進(jìn)腫瘤細(xì)胞的增殖［16］。除此之外，熱休克蛋白(HSP70和HSP90)和survivan蛋白因腫瘤細(xì)胞缺氧而增加。以上這些蛋白均可抑制細(xì)胞凋亡、促進(jìn)腫瘤細(xì)胞增殖，進(jìn)而對(duì)促進(jìn)腫瘤細(xì)胞生長(zhǎng)的微環(huán)境提供強(qiáng)大的刺激作用［17-18］。一方面，外泌體可以將自身的細(xì)胞因子釋放至細(xì)胞外，作用于內(nèi)皮細(xì)胞膜表面受體，間接促使腫瘤新生血管的形成。富含TGFβ1蛋白的腫瘤源性外泌體將靜息狀態(tài)的成纖維細(xì)胞活化成肌成纖維細(xì)胞，從而促進(jìn)腫瘤血管新生和腫瘤生長(zhǎng)，而單純的可溶性TGFβ1雖然可以激活成纖維細(xì)胞，但卻沒(méi)有促進(jìn)腫瘤血管新生和腫瘤生長(zhǎng)的作用［19-20］。另一方面，腫瘤細(xì)胞來(lái)源的外泌體可傳遞組織因子、miRNA-210等直接調(diào)控血管內(nèi)皮細(xì)胞的靶基因或信號(hào)通路，促進(jìn)腫瘤血管新生，為腫瘤的侵襲轉(zhuǎn)移創(chuàng)造條件［21］。人體免疫系統(tǒng)正常功能的發(fā)揮可以減少腫瘤的進(jìn)展，然而有研究表明，腫瘤患者存在免疫抑制，進(jìn)而進(jìn)一步導(dǎo)致腫瘤的快速生長(zhǎng)、進(jìn)展和播散［22］，尤其是從相對(duì)應(yīng)惡性病變部位分離出的外泌體會(huì)促進(jìn)腫瘤細(xì)胞的快速生長(zhǎng)［16］。外泌體可通過(guò)增加免疫抑制細(xì)胞、減少NK細(xì)胞和T細(xì)胞的增殖和細(xì)胞毒性、減少抗原遞呈細(xì)胞的數(shù)量和功能等來(lái)抑制免疫，說(shuō)明腫瘤細(xì)胞源性的外泌體可介導(dǎo)病變部位導(dǎo)致的免疫抑制［23-26］。如胃癌細(xì)胞釋放的外泌體可以向抗腫瘤的T細(xì)胞傳遞凋亡信號(hào)，而不需要直接的細(xì)胞-細(xì)胞接觸。這些外泌體能部分地被誘導(dǎo)上調(diào)Cbl-b和c-Cbl，抑制Akt的活性及caspase-3、caspase-8和caspase-9的激活。外泌體與免疫細(xì)胞之間的相互作用揭示了腫瘤抑制免疫功能的潛在機(jī)制［27］。

2 外泌體源性非編碼RNA與消化系統(tǒng)惡性腫瘤

2.1 外泌體源性miRNA與消化系統(tǒng)惡性腫瘤

外泌體中富含miRNA。成熟miRNA占外泌體所有RNA的40%以上，由起源細(xì)胞分泌，發(fā)揮沉默靶細(xì)胞的靶mRNA的作用［28］。外泌體源性miRNA通過(guò)介導(dǎo)細(xì)胞通訊來(lái)調(diào)節(jié)基因表達(dá)［29］。

外泌體源性miRNA通過(guò)腫瘤內(nèi)皮細(xì)胞、血管生成、細(xì)胞通訊和免疫抑制等介導(dǎo)腫瘤發(fā)生、發(fā)展的過(guò)程。Liao等［30］研究報(bào)道，食管癌細(xì)胞外泌體源性的miR-21可以通過(guò)調(diào)控抑癌基因PDCD4和激活其下游的JNK信號(hào)通路，促進(jìn)腫瘤細(xì)胞的進(jìn)展、轉(zhuǎn)移。Zhou等［31］研究發(fā)現(xiàn)，胰腺癌細(xì)胞來(lái)源的外泌體可通過(guò)miR-203下調(diào)樹(shù)突細(xì)胞TLR4的表達(dá)，進(jìn)而引起免疫抑制導(dǎo)致胰腺癌的發(fā)生。Que等［32］研究發(fā)現(xiàn)，胰腺癌外泌體源性miRNA可下調(diào)DC/細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞(cytokine-induced killer cells，CIKs)的抗腫瘤活性，其水平的降低可增強(qiáng)DC/CIKs的抗腫瘤活性。有研究發(fā)現(xiàn)，有11種miRNA特異的存在于Hep3B細(xì)胞來(lái)源的外泌體中，說(shuō)明肝細(xì)胞癌的外泌體可選擇性募集一系列特異表達(dá)的miRNA［33］。肝細(xì)胞癌的癌細(xì)胞外泌體源性miRNA可通過(guò)抑制TAK1及與TAK1相關(guān)的下游信號(hào)通路，增強(qiáng)受體細(xì)胞中轉(zhuǎn)化細(xì)胞的生長(zhǎng)，進(jìn)而導(dǎo)致肝細(xì)胞癌的發(fā)展［34］。

2.1.1 外泌體源性miRNA與消化系統(tǒng)惡性腫瘤的早期診斷

消化系統(tǒng)惡性腫瘤通常在診斷時(shí)已是局部晚期或發(fā)生轉(zhuǎn)移，錯(cuò)過(guò)最佳治療時(shí)機(jī)，因此提高消化系統(tǒng)惡性腫瘤早期診斷率仍是亟待解決的問(wèn)題。不少研究以外泌體源性miRNA作為切入點(diǎn)，探討其作為早期診斷指標(biāo)的可能性，并取得較好的研究結(jié)果。Machida等［35］研究發(fā)現(xiàn)，和健康對(duì)照組相比，胰膽道惡性腫瘤患者唾液外泌體源性的miRNA-1246和miRNA-4644顯著升高，且兩者聯(lián)合檢測(cè)，受試者工作特征(receiver operating characteristic curve，ROC)曲線的曲線下面積達(dá)到0.833，說(shuō)明胰膽道惡性腫瘤患者唾液外泌體源性的miRNA可作為早期診斷的標(biāo)志物。Madhavan等［36］研究發(fā)現(xiàn)，miRNA-1246、miRNA-4644、miRNA-3976及miRNA-4306在83%的胰腺癌患者血清外泌體中顯著升高，而在胰腺良性腫瘤患者及健康對(duì)照組中沒(méi)有明顯變化，提示胰腺腫瘤細(xì)胞來(lái)源的miRNA的升高具有診斷價(jià)值。Wang等［37］研究發(fā)現(xiàn)，肝細(xì)胞癌血清外泌體源性miRNA-21的表達(dá)顯著高于慢性乙型肝炎和健康對(duì)照者，且其敏感性較高，說(shuō)明外泌體源性miRNA-21可作為早期診斷肝細(xì)胞癌的潛在生物學(xué)指標(biāo)。

2.1.2 外泌體源性miRNA與消化系統(tǒng)惡性腫瘤的轉(zhuǎn)移、侵襲及預(yù)后評(píng)估

腫瘤細(xì)胞來(lái)源的外泌體影響腫瘤的侵襲、轉(zhuǎn)移。一方面，外泌體可作為載體將miRNA從原發(fā)部位運(yùn)輸?shù)狡渌课?，促進(jìn)腫瘤的轉(zhuǎn)移；另一方面，通過(guò)轉(zhuǎn)運(yùn)具有侵襲、轉(zhuǎn)移能力的miRNA，提高腫瘤的復(fù)發(fā)、轉(zhuǎn)移潛能。Tanaka等［38］研究發(fā)現(xiàn)，食管鱗狀細(xì)胞癌患者血清的外泌體能誘導(dǎo)食管鱗狀細(xì)胞癌細(xì)胞的增殖。與食管良性腫瘤患者相比，外泌體源性的miR-21在食管鱗狀細(xì)胞癌的患者血清中高表達(dá)，并且和腫瘤的進(jìn)展及侵襲相關(guān)指標(biāo)呈正相關(guān)。而沒(méi)有外泌體的血清中未能檢測(cè)到miR-21，說(shuō)明和腫瘤相關(guān)的miRNA可能特異的存在于外泌體中，并通過(guò)外泌體的轉(zhuǎn)運(yùn)功能發(fā)揮作用。Nishida等［39］研究發(fā)現(xiàn)，miR-10b在結(jié)直腸癌細(xì)胞外泌體中顯著高表達(dá)，且miR-10b的高表達(dá)和淋巴結(jié)轉(zhuǎn)移及差的預(yù)后相關(guān)。miR-122是肝臟特異的抗細(xì)胞增殖的miRNA，可通過(guò)人類肝細(xì)胞癌的癌細(xì)胞的外泌體進(jìn)行轉(zhuǎn)運(yùn)。miR-122的缺失或下調(diào)導(dǎo)致肝細(xì)胞癌的進(jìn)展，并且與預(yù)后差及轉(zhuǎn)移密切相關(guān)［40-42］。

2.2 外泌體源性lncRNA與消化系統(tǒng)惡性腫瘤

外泌體中的lncRNA約占外泌體總RNA的3%，其介導(dǎo)腫瘤發(fā)生的認(rèn)識(shí)體現(xiàn)在：某些特異具有生物學(xué)功能的lncRNA可被選擇性載入外泌體中；外泌體可轉(zhuǎn)運(yùn)具有直接沉默表觀遺傳作用的lncRNA；腫瘤細(xì)胞釋放富含lncRNA的外泌體，誘導(dǎo)受體細(xì)胞發(fā)生腫瘤樣表型的改變［43-44］。

2.2.1 外泌體源性lncRNA與消化系統(tǒng)惡性腫瘤發(fā)生、發(fā)展的關(guān)系

近期研究顯示，外泌體可作為功能性lncRNA的轉(zhuǎn)運(yùn)小泡，對(duì)受體細(xì)胞的表型產(chǎn)生作用。LncRNA TUC339存在于肝細(xì)胞癌的癌細(xì)胞外泌體中，并且在外泌體中高表達(dá)。抑制這種lncRNA的表達(dá)可減少細(xì)胞增殖、克隆細(xì)胞的生長(zhǎng)及細(xì)胞黏附，說(shuō)明細(xì)胞利用外泌體及TUC339增加周圍細(xì)胞的增殖，進(jìn)而促進(jìn)肝細(xì)胞癌的發(fā)展［45］。外泌體源性lncRNA通過(guò)調(diào)節(jié)腫瘤微環(huán)境促進(jìn)腫瘤的發(fā)生、發(fā)展。有研究顯示，肝細(xì)胞癌CD90+Huh7細(xì)胞可分泌包含有多種lncRNA(HOTAIR、HULU、linc-ROR和H19)的外泌體，與該細(xì)胞分泌的外泌體在試管內(nèi)共培養(yǎng)，外泌體被內(nèi)皮細(xì)胞迅速內(nèi)化，促使細(xì)胞重組為管狀樣結(jié)構(gòu)，并上調(diào)血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor，VEGF)/ VEGF-R1 mRNA的表達(dá)水平，導(dǎo)致促血管生成作用，且使細(xì)胞黏附分子增加，參與內(nèi)皮細(xì)胞的外滲過(guò)程，促進(jìn)腫瘤的進(jìn)展［46］。

2.2.2 外泌體源性lncRNA在消化系統(tǒng)惡性腫瘤臨床診治中的潛在應(yīng)用價(jià)值

類似于miRNA，外泌體源性lncRNA廣泛存在于多種體液中，樣品取材方便，性質(zhì)穩(wěn)定，可通過(guò)常用實(shí)驗(yàn)技術(shù)如實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(real-time fluorescent quantitative polymerase chain reaction，RTFQ-PCR)、基因芯片及測(cè)序等檢測(cè)，具有較好的臨床應(yīng)用前景［15,47-48］。

LncRNA穩(wěn)定存在于血漿、血清和其他體液中，可作為非侵入性的指標(biāo)成為腫瘤診斷的標(biāo)志物［49-50］。與胃上皮不典型增生患者和健康對(duì)照組相比，LINC00152在胃癌患者血漿外泌體中高表達(dá)，并且LINC00152的表達(dá)水平在等量血漿及等量血漿來(lái)源的外泌體中差異無(wú)統(tǒng)計(jì)學(xué)意義，提示LINC00152在血漿中受外泌體的保護(hù)而穩(wěn)定存在于外泌體中。外泌體源性LINC00152診斷胃癌的靈敏度是48.1%，特異度是85.2%，ROC曲線的曲線下面積是0.66，提示其具有較好的診斷優(yōu)勢(shì)［51］。此外，血清外泌體源性lncRNA-CRNDE-h的升高可以鑒別結(jié)直腸癌及結(jié)直腸良性疾病患者、健康對(duì)照者，其診斷的靈敏度和特異度分別為70.3%和94.4%，ROC曲線的曲線下面積達(dá)0.89，可用于結(jié)直腸癌患者的早期診斷［52］。

外泌體源性lncRNA在一定程度上影響著消化系統(tǒng)腫瘤的轉(zhuǎn)移、侵襲及預(yù)后。ROR是一種lncRNA，在阿霉素處理過(guò)的肝細(xì)胞癌的癌細(xì)胞的外泌體中高表達(dá)，經(jīng)含有ROR的外泌體處理肝細(xì)胞癌的癌細(xì)胞后，發(fā)現(xiàn)其化療抵抗作用增強(qiáng)，敲除ROR后化療的靈敏度增加。說(shuō)明腫瘤細(xì)胞可通過(guò)外泌體及l(fā)ncRNA來(lái)增加周圍細(xì)胞的化療抵抗，促進(jìn)腫瘤的局部侵襲能力［53］。TGF-β可誘導(dǎo)上皮細(xì)胞間質(zhì)化，促進(jìn)癌細(xì)胞的侵襲和轉(zhuǎn)移。TGF-β誘導(dǎo)胰腺癌細(xì)胞中的多個(gè)lncRNA上調(diào)，其中HULU的變化最為明顯。沉默HULU后細(xì)胞的存活、侵襲和遷移能力減弱，經(jīng)TGF-β誘導(dǎo)胰腺癌細(xì)胞來(lái)源外泌體中的HULU水平也增高。經(jīng)癌細(xì)胞及Panc-1胰腺癌細(xì)胞來(lái)源的外泌體溫育后，細(xì)胞中的黏附分子增加，說(shuō)明胰腺癌細(xì)胞可通過(guò)外泌體源性的lncRNA-HULU來(lái)增強(qiáng)細(xì)胞的侵襲及遷移能力［54］。結(jié)直腸癌患者血清外泌體源性lncRNA-CRNDE-h的水平與局部淋巴結(jié)轉(zhuǎn)移(P=0.019)和遠(yuǎn)處轉(zhuǎn)移相關(guān)(P=0.003)，可用于評(píng)價(jià)結(jié)直腸癌患者的轉(zhuǎn)移情況及預(yù)后［52］。

2.3 外泌體源性circRNA與消化系統(tǒng)惡性腫瘤

2.3.1 CircRNA概述

CircRNA是一類新發(fā)現(xiàn)的非編碼RNA分子，由一個(gè)或幾個(gè)外顯子形成的環(huán)狀結(jié)構(gòu)［55］。CircRNA既可以與miRNA結(jié)合，通過(guò)影響miRNA下游靶基因來(lái)發(fā)揮調(diào)控作用，又可以調(diào)控選擇性剪接、宿主基因轉(zhuǎn)錄［56］。最早于20世紀(jì)70年代在病毒中發(fā)現(xiàn)，后來(lái)發(fā)現(xiàn)在真核細(xì)胞中也存在［57-58］。目前隨著高通量測(cè)序等技術(shù)的快速發(fā)展，circRNA目前被證實(shí)在真核細(xì)胞轉(zhuǎn)錄組中大量存在［59-62］。具有種類多樣、存在普遍、序列保守、不被核酸外切酶降解、組織和時(shí)序特異性的生物學(xué)特點(diǎn)［63］。關(guān)于其生物合成的機(jī)制主要有外顯子跳讀導(dǎo)致成環(huán)、反向剪接成環(huán)及蛋白促進(jìn)成環(huán)［64］。

2.3.2 外泌體源性circRNA與消化系統(tǒng)惡性腫瘤

Li等［65］首次報(bào)道，不僅細(xì)胞來(lái)源的外泌體，人血清外泌體中也含有大量完整穩(wěn)定的circRNA，并且結(jié)直腸癌患者血清外泌體中的circ-KLHDC10與健康人群差異明顯，提示腫瘤來(lái)源的exo-circRNA有望成為腫瘤檢測(cè)的潛在標(biāo)志物。目前，外泌體源性circRNA在消化系統(tǒng)惡性腫瘤發(fā)生、發(fā)展過(guò)程中的分子機(jī)制、臨床應(yīng)用等認(rèn)識(shí)還很有限，需要進(jìn)一步深入的研究。

3 小結(jié)與展望

外泌體及外泌體源性非編碼RNA在腫瘤發(fā)生、發(fā)展中發(fā)揮重要作用，可為多種疾病的發(fā)生機(jī)制提供思路，也為臨床診斷、預(yù)后及治療提供新的標(biāo)志物。但目前關(guān)于外泌體及外泌體源性非編碼RNA的研究仍處于初始階段，面臨缺乏完善的數(shù)據(jù)庫(kù)、機(jī)制不明確及腫瘤特異性較強(qiáng)的非編碼RNA不多等問(wèn)題。相信隨著生物技術(shù)的不斷發(fā)展，外泌體及外泌體源性非編碼RNA在腫瘤中的功能及機(jī)制會(huì)逐漸被發(fā)現(xiàn)，為腫瘤的診治提供有效依據(jù)。

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Research progress on exosome-derived non-coding RNA in digestive system malignancy

YU Shulin, CHEN Zhen (Department of Integrative Oncology, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China)

CHEN Zhen E-mail: zhenczl@fudan.edu.cn

Exosome is a kind of biological membrane structure at nanometer level. It is secreted by various cells and widely distributed in most body fluids. Biological active substances, including non-coding RNAs and proteins, have been identified in the exosomes. Exosome may be involved in cell-to-cell substance transporting and signaling. Non-coding RNAs have important function of regulating gene expression and also play key roles in the occurrence and development of many cancers. Research on exosome-derived non-coding RNA is a new hotspot at present. In this paper, we will review research progress on exosome and exosome-derived non-coding RNA in digestive system malignancy.

Exosome; Non-coding RNAs; Digestive system malignancy

10.19401/j.cnki.1007-3639.2017.04.010

R735

A

1007-3639(2017)04-0297-07

2016-12-19

2017-03-06）

國(guó)家自然科學(xué)基金資助項(xiàng)目(81573752)。

陳 震 E-mail: zhenczl@fudan.edu.cn