丙型肝炎病毒誘發(fā)肝細(xì)胞肝癌的分子機制

高榮，徐力，徐杰，任浩

第二軍醫(yī)大學(xué)微生物學(xué)教研室，上海市醫(yī)學(xué)生物防護(hù)重點實驗室，上海 200433

·綜述·

丙型肝炎病毒誘發(fā)肝細(xì)胞肝癌的分子機制

高榮，徐力，徐杰，任浩

第二軍醫(yī)大學(xué)微生物學(xué)教研室，上海市醫(yī)學(xué)生物防護(hù)重點實驗室，上海 200433

肝細(xì)胞肝癌(hepatocellular carcinoma，HCC)是影響人類健康的惡性腫瘤之一，丙型肝炎病毒(hepatitis C virus，HCV)是其主要致病因素之一。HCV誘發(fā)的HCC是由病毒和宿主免疫介導(dǎo)的多步驟復(fù)雜過程，從慢性炎癥發(fā)展到肝硬化和肝癌，病毒和宿主因子共同參與此過程。其中，宿主基因突變是導(dǎo)致HCC發(fā)生的危險因素之一，全面了解HCV誘發(fā)HCC的分子機制將有助于解決此問題。

丙型肝炎病毒；肝細(xì)胞肝癌；分子機制

肝細(xì)胞肝癌(hepatocellular carcinoma，HCC)是人類最常見的惡性腫瘤之一，在男性疾病中居第5位，在女性疾病中居第9位，已成為全球癌癥相關(guān)死亡的第二大病因。在多數(shù)西方國家，丙型肝炎病毒(hepatitis C virus，HCV)是慢性肝病和HCC的主要危險因素[1]。全球約3%的人口感染HCV，大部分感染者不能清除病毒感染并發(fā)展為慢性丙型肝炎(chronic hepatitis C，CHC)。CHC可引起進(jìn)行性肝纖維化相關(guān)肝損傷，20～40年后可能演變?yōu)楦斡不?10%～20%)和HCC(1%～5%)[2-3]。據(jù)估計，全球27%的肝硬化和25%的HCC可歸因于HCV感染[4]。慢性HCV感染患者發(fā)生HCC的危險與肝纖維化階段緊密相關(guān)，肝硬化患者的HCC發(fā)病率遠(yuǎn)高于輕度纖維化患者[5]。此外，其他危險因素，如乙型肝炎病毒(hepatitis B virus，HBV)/人類免疫缺陷病毒(human immunodeficiency virus，HIV)共感染、肥胖、胰島素抵抗、非酒精性脂肪性肝炎等，會加快HCV誘發(fā)的HCC (HCV-induced HCC，HCV-HCC)進(jìn)程[6]?；诟蓴_素治療的持續(xù)病毒學(xué)應(yīng)答(sustained virological response，SVR)可降低大部分HCV患者的HCC發(fā)生率，表明清除病毒在阻止腫瘤發(fā)生中的重要性[7]。將直接抗病毒藥物(direct-acting antiviral agent，DAA)引入抗HCV治療，可明顯提高SVR。然而，DAA在降低HCV相關(guān)肝硬化患者HCC發(fā)生率及促進(jìn)HCC復(fù)發(fā)方面仍有爭議。有報道表明，DAA治療可促進(jìn)HCV-HCC患者病情惡化，且更難以治療[8]。因此，闡明HCV-HCC分子機制有助于探討HCC預(yù)防和治療新策略，本文主要就此進(jìn)行簡要綜述。

1 HCV的基因組結(jié)構(gòu)與功能

HCV為黃病毒屬單股正鏈RNA病毒，基因組全長約9.6 kb，具有單一的開放讀碼框，編碼長約 3 000 個氨基酸殘基的多蛋白前體。5′和3′端均具有高度保守的非編碼區(qū)(non-coding region，NCR)，這些序列對病毒蛋白的合成及復(fù)制是必需的。多蛋白前體在宿主和病毒蛋白酶作用下形成的結(jié)構(gòu)蛋白包括核心(Core)蛋白、包膜蛋白E1和E2，其序列位于近5′區(qū)；非結(jié)構(gòu)蛋白包括NS2～NS5，其序列位于近3′區(qū)[9]。Core蛋白序列保守，除保護(hù)HCV核心結(jié)構(gòu)外，還具有促進(jìn)HCV復(fù)制、調(diào)節(jié)宿主免疫細(xì)胞等功能[10]。包膜蛋白E1和E2均定位于內(nèi)質(zhì)網(wǎng)并形成異二聚體，與病毒入侵和病毒顆粒組裝有關(guān)。E2蛋白還具有高度變異性，參與病毒的免疫逃逸[11]。NS2是一類跨膜蛋白，作用類似金屬蛋白酶，除剪切 NS3外，還參與NS5A磷酸化[12]。NS3分為2個結(jié)構(gòu)域，N端的181個氨基酸具有蛋白酶活性，C端的465個氨基酸具有螺旋酶及NTP酶活性，后兩者參與病毒復(fù)制[13]。NS4A是NS3輔助因子，參與其催化反應(yīng)，而NS4B是NS5A磷酸化反應(yīng)的輔助因子[14]。NS5A可能通過與雙鏈RNA依賴的蛋白激酶(double-stranded RNA-dependent protein kinase，PKR)結(jié)合，使病毒對干擾素產(chǎn)生耐藥，并參與HCV多種蛋白的成熟和RNA復(fù)制，調(diào)控宿主多種基因表達(dá)，刺激細(xì)胞增殖，抑制細(xì)胞凋亡及影響干擾素療效[15]。NS5B編碼RNA依賴的RNA聚合酶(RNA-dependent RNA polymerase，RdRp)，作為核心酶在HCV復(fù)制中起重要作用[16]。

2 HCV感染與HCC發(fā)生

HCV感染所致肝癌是一個由病毒、宿主和環(huán)境等多方因素共同參與的復(fù)雜過程。除通過免疫介導(dǎo)的慢性炎癥反應(yīng)間接誘發(fā)肝癌外，還可能通過改變宿主的信號通路(如增殖、能量代謝、血管生成、上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition，EMT)、DNA修復(fù)、細(xì)胞凋亡和氧化/內(nèi)質(zhì)網(wǎng)應(yīng)激等)直接誘發(fā)HCC[17]。慢性炎癥可在代謝和基因水平損傷肝細(xì)胞并使導(dǎo)致細(xì)胞死亡的活性氧(reactive oxygen species，ROS)水平增加。HCV感染后的肝細(xì)胞再生可使染色體不穩(wěn)定性增加，并促進(jìn)肝細(xì)胞新陳代謝，以及惡性克隆進(jìn)展的遺傳/表觀遺傳發(fā)生不可逆的變異[18]。

2.1 HCV直接干預(yù)致癌和抗癌通路

2.1.1HCV與細(xì)胞周期HCV蛋白可直接作用于某些宿主腫瘤抑制基因和原癌基因[19]。例如，HCV NS5B可結(jié)合視網(wǎng)膜母細(xì)胞瘤(retinoblastoma，Rb)蛋白并促使其在胞質(zhì)重新定位和蛋白酶體降解，最終激活促進(jìn)細(xì)胞周期進(jìn)展的E2F(細(xì)胞分裂G1期進(jìn)入S期的重要轉(zhuǎn)錄因子)基因應(yīng)答[19]。此外，HCV蛋白還可激活Cyclin/Cdk復(fù)合物來促進(jìn)G1/S轉(zhuǎn)換，進(jìn)而干擾細(xì)胞周期進(jìn)程。如NS2蛋白能激活Cyclin D/Cdk4并上調(diào)Cyclin E表達(dá)，而Core蛋白上調(diào)Cyclin E和Cdk2表達(dá)[20-22]。

2.1.2HCV與生長因子信號通路生長因子信號通路激活對HCC的發(fā)生和維持起重要作用。HCV Core、E2、NS3和NS5A蛋白通過干擾Raf/絲裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)/細(xì)胞外調(diào)節(jié)蛋白激酶(extracellular regulated protein kinase，ERK)通路刺激細(xì)胞增殖[23-26]。HCV也可激活Wnt/β-catenin通路，通過Wnt配體與Frizzled受體結(jié)合活化，阻止β-catenin多聚蛋白復(fù)合體形成，從而激活c-myc和Cyclin D等一系列靶基因的轉(zhuǎn)錄[27]。體外實驗表明，HCV NS5A和Core過表達(dá)可激活β-catenin，其間接機制為抑制GSK-3β激活及使β-catenin穩(wěn)定化。最近研究表明，β-catenin可與NS5A蛋白相互作用而被直接激活[28]。目前為止，HCV感染細(xì)胞內(nèi)β-catenin活化的功能意義尚不清楚。雖然Wnt信號通路失調(diào)不足以引起肝細(xì)胞惡性轉(zhuǎn)化，但β-catenin表達(dá)水平在多數(shù)HCC患者中升高，提示其可能參與腫瘤生長的維持[29]。

2.1.3HCV與轉(zhuǎn)化因子信號通路轉(zhuǎn)化生長因子β(transforming growth factor β，TGF-β)是一種多向性和多效性的細(xì)胞因子，可通過細(xì)胞表面受體信號轉(zhuǎn)導(dǎo)途徑發(fā)揮抗增殖、促凋亡作用，在EMT過程中具有促腫瘤活性[30]。HCV NS5A蛋白與TGF-β受體1(transforming growth factor β receptor 1，TGFBR1)結(jié)合抑制TGF-β介導(dǎo)的Smad2磷酸化和核易位及Smad3/Smad4異源二聚體形成，從而阻斷TGF-β信號轉(zhuǎn)導(dǎo)[31]。此外，TGF-β通路也是HCV Core蛋白的作用靶點[32]。值得注意的是，與經(jīng)典的TGFBR1通路不同，由c-Jun氨基末端激酶(c-Jun N-terminal kinase，JNK)介導(dǎo)的Smad3蛋白磷酸化可通過上調(diào)纖溶酶原激活物抑制劑1(plasminogen activator inhibitor 1，PAI-1)促進(jìn)細(xì)胞外基質(zhì)沉積，并在慢性肝病-肝硬化-HCC這一疾病演變過程中表達(dá)[33]。此外，HCV可間接通過激活磷脂酰肌醇-3激酶(phosphoinositide-3 kinase，PI3K)/蛋白激酶B(protein kinase B，Akt)通路抑制細(xì)胞凋亡，這種作用可能是通過NS5A下調(diào)PTEN并減弱PI3K/Akt通路的抑制作用實現(xiàn)的[34]。

2.1.4HCV與細(xì)胞凋亡細(xì)胞凋亡是為維持內(nèi)環(huán)境穩(wěn)定，由基因控制的細(xì)胞自主有序的死亡，是一個主動過程。凋亡抑制是腫瘤發(fā)生多步驟過程中的重要事件，主要表現(xiàn)為惡性轉(zhuǎn)化細(xì)胞在細(xì)胞周期中逃避關(guān)鍵檢查點。HCV感染與凋亡之間的相互作用尚未完全闡明。細(xì)胞實驗中，細(xì)胞凋亡隨時間變化，凋亡活性在感染早期較高，并隨時間減少[35]。過度凋亡在急性肝炎、急性重型肝炎及慢性肝炎患者中均可觀察到。持續(xù)凋亡與持續(xù)性炎癥和纖維化有關(guān)，而缺乏細(xì)胞凋亡則有助于HCC的發(fā)生[36]。

P53是一個關(guān)鍵的腫瘤抑制蛋白，是細(xì)胞生長周期的負(fù)調(diào)控因子，可阻滯細(xì)胞周期，促進(jìn)細(xì)胞凋亡。HCV Core蛋白可直接激活P53信號通路，但這種相互作用仍有爭議[37]。此外，HCV NS2、NS3/4A和NS5A可誘導(dǎo)P53蛋白從細(xì)胞核到細(xì)胞質(zhì)/核周區(qū)域重新定位從而干擾P53通路[22]。HCV Core蛋白通過誘導(dǎo)FLICE阻斷腫瘤壞死因子α(tumor necrosis factor α，TNF-α)介導(dǎo)的凋亡信號，HCV NS5A通過阻斷天冬氨酸特異性半胱氨酸蛋白酶3(cysteinyl aspartate specific proteinase 3，caspase-3)和抑制多聚 ADP核糖聚合酶1〔poly (ADP-ribose) polymerase 1，PARP1〕，防止TNF-α介導(dǎo)細(xì)胞凋亡[38]。

2.1.5HCV與自噬自噬又稱Ⅱ型細(xì)胞死亡，是細(xì)胞在自噬相關(guān)基因調(diào)控下，利用溶酶體降解自身受損的細(xì)胞器和大分子物質(zhì)的過程。HCV可抑制宿主細(xì)胞通過自噬對病毒的清除，從而起促進(jìn)腫瘤形成的作用[39]。自噬缺陷性小鼠可引起HCC自發(fā)形成，這主要是由于P62蛋白堆積和隨后轉(zhuǎn)錄因子Nrf2(可調(diào)節(jié)多種細(xì)胞保護(hù)基因)的持續(xù)激活。P62水平在HCV-HCC患者體內(nèi)增高，提示自噬損傷在腫瘤發(fā)展中可能具有重要作用[40]。此外，自噬缺乏的肝細(xì)胞也可促進(jìn)EMT[41]。另一方面，在抗肝細(xì)胞大鼠模型中，自噬可促進(jìn)癌前病變的肝臟結(jié)節(jié)生長[42]?？傊?，HCV感染引發(fā)的自噬在病毒復(fù)制/分泌、炎癥和脂質(zhì)代謝平衡等方面發(fā)揮重要作用。

2.2 HCV誘導(dǎo)的免疫反應(yīng)與HCC

HCV通過多種機制(抑制Ⅰ型IFN產(chǎn)生，改變CD4+T細(xì)胞向Th2細(xì)胞、Th17細(xì)胞、調(diào)節(jié)性T細(xì)胞分化等)逃避宿主先天性免疫和獲得性免疫而建立慢性感染[46-47]。這些改變導(dǎo)致肝臟輕度慢性炎癥，并誘導(dǎo)ROS和一氧化氮釋放，增強脂質(zhì)過氧化作用及毒性細(xì)胞因子的異常表達(dá)，同時破壞宿主免疫監(jiān)視而促進(jìn)肝癌發(fā)展[48]。

TNF-α、白細(xì)胞介素1(interleukin 1，IL-1)、IL-23和IL-6、淋巴毒素α(lymphotoxin α，LTα)和LT β等多種炎性細(xì)胞因子參與慢性肝炎和HCC的發(fā)生和發(fā)展。其中，LTα和LTβ在HCC發(fā)展中起重要作用，LT高表達(dá)誘導(dǎo)的嚴(yán)重肝損傷與肝浸潤淋巴細(xì)胞(liver infiltrating lymphocyte，LIL)數(shù)量增加相關(guān)[49]。HCV感染期間，喪失病毒清除作用的LIL在肝臟中積累并建立慢性炎癥。值得注意的是，Ramzan等發(fā)現(xiàn)，與無HCC的HCV患者相比，HCV-HCC患者的肝硬化組織中存在較高水平的肝浸潤性T和B細(xì)胞。特別是大量存在于肝硬化區(qū)域而不是HCV-HCC患者腫瘤組織中的CD8+T細(xì)胞與HCC發(fā)生相關(guān)，且是手術(shù)切除后復(fù)發(fā)的預(yù)后因素[50]。此外，在HCV-HCC患者中，炎癥性CD8+T細(xì)胞的增加伴隨著參與腫瘤免疫監(jiān)視的自然殺傷(natural killer，NK)細(xì)胞和自然殺傷T(natural killer T，NKT)細(xì)胞的減少[50]。相反，具有抗腫瘤免疫關(guān)鍵作用的調(diào)節(jié)性T細(xì)胞在HCC和HCV-肝硬化組織中增加[51]。這些數(shù)據(jù)表明，在HCV到肝硬化再到HCC的發(fā)展過程中，腫瘤促進(jìn)炎癥反應(yīng)和抗腫瘤免疫受損的協(xié)同效應(yīng)可能至關(guān)重要。

2.3 HCV誘導(dǎo)的脂質(zhì)代謝異常與HCC

HCV-HCC常伴有脂肪性肝炎，表明與炎癥相關(guān)的脂質(zhì)代謝改變可參與癌癥發(fā)生[52]。HCV蛋白主要通過活化脂肪生成途徑和降低脂質(zhì)分解代謝干擾宿主脂質(zhì)代謝，從而觸發(fā)感染肝細(xì)胞中的脂毒性[53]。慢性HCV患者應(yīng)用他汀類藥物可使伴隨的肝硬化和HCC呈劑量依賴性減少，證實脂肪變性與HCC關(guān)系密切[54]。近期有研究提出HCV和肥胖可協(xié)同誘導(dǎo)HCC的新機制。NS5A通過增加Toll樣受體4(Toll-like receptor 4，TLR4)水平，增強干細(xì)胞轉(zhuǎn)錄因子NANOG的誘導(dǎo)作用，從而產(chǎn)生腫瘤起始干細(xì)胞樣細(xì)胞(tumor-initiating stem-like cell，TIC)[55]。此外，干細(xì)胞轉(zhuǎn)錄因子NANOG還可誘導(dǎo)EMT主調(diào)節(jié)器Twist1協(xié)同瘦素(leptin)-STAT3通路促進(jìn)腫瘤發(fā)生，活化脂肪酸氧化，支持TIC自我更新和耐藥性產(chǎn)生[56]。

HCV誘導(dǎo)脂肪變性促進(jìn)HCC發(fā)展的一個重要方面是改變肝臟T細(xì)胞功能。最近，在脂肪性肝炎和肥胖復(fù)合小鼠模型中觀察到大量CD8+T和NKT細(xì)胞肝浸潤，它們的激活可誘導(dǎo)肝細(xì)胞脂肪變性，進(jìn)而發(fā)揮炎癥和致癌作用[57]。在肥胖誘導(dǎo)小鼠模型中，脂質(zhì)生成增加(特別是亞油酸)與選擇性CD4+T細(xì)胞丟失直接相關(guān)，而選擇性CD4+T細(xì)胞可削弱抗腫瘤免疫而促進(jìn)HCC進(jìn)展[58]。

HCV患者的HCC發(fā)病風(fēng)險也與肝纖維化程度緊密聯(lián)系。促纖維形成細(xì)胞因子已被鑒定為HCC進(jìn)展的關(guān)鍵調(diào)節(jié)劑。TGF-β是參與纖維形成的關(guān)鍵細(xì)胞因子，直接由HCV Core蛋白或氧化、內(nèi)質(zhì)網(wǎng)應(yīng)激和核因子κB(nuclear factor κB，NF-κB)通路活化誘導(dǎo)產(chǎn)生[59]。最近，Jee等證實HCV患者肝細(xì)胞表達(dá)高水平TGF-β，且體外TGF-β的分泌量足以活化肝星狀細(xì)胞[60]。刺激后的肝星狀細(xì)胞在肌成纖維細(xì)胞中轉(zhuǎn)分化并釋放促纖維形成介質(zhì)、炎性細(xì)胞因子和趨化因子，加重肝臟炎癥和纖維化[61]。TGF-β除具有促纖維形成作用，還是免疫抑制劑，有利于腫瘤轉(zhuǎn)化細(xì)胞的免疫逃逸[62]。

2.4 HCV誘導(dǎo)的促血管生成與HCC

新血管生成是癌細(xì)胞生長和存活的必要階段。許多研究表明，所有HCC患者中，HCV陽性者微血管密度較高[63]。HCV結(jié)構(gòu)和非結(jié)構(gòu)蛋白在此過程中發(fā)揮直接作用。HCV Core蛋白通過上調(diào)低氧誘導(dǎo)因子1α(hypoxia inducible factor 1α，HIF-1α)促進(jìn)血管生成，HIF-1α可上調(diào)血管內(nèi)皮生長因子(vascular endothelial growth factor，VEGF)和環(huán)加氧酶2，也可激活基質(zhì)金屬蛋白酶(matrix metalloproteinase，MMP)如MMP-2和MMP-9[64]。VEGF是HCC中重要的內(nèi)皮特異性生長因子，可作為HCC的預(yù)后因子[65]。HCV感染上調(diào)促血管生成素2(angiopoietin 2，Ang-2)水平，Ang-2可與Tie-1受體結(jié)合抑制血管生成。在VEGF刺激下，Ang-2可從內(nèi)皮細(xì)胞釋放并促進(jìn)VEGF的促血管生成作用[66]。

EMT在癌細(xì)胞轉(zhuǎn)移過程中發(fā)揮核心作用，此過程中上皮細(xì)胞獲得間充質(zhì)細(xì)胞特征，失去細(xì)胞極性，失去與基底膜的連接，從而使細(xì)胞運動性和侵襲性增加[67]。值得注意的是，NS5A通過激活Twist2觸發(fā)EMT，而Twist2是轉(zhuǎn)移過程中的關(guān)鍵轉(zhuǎn)錄調(diào)節(jié)子[68]。另一種EMT誘導(dǎo)途徑由E1/E2蛋白觸發(fā)，并通過TGF-β和VEGF信號通路轉(zhuǎn)導(dǎo)。TGF-β和VEGF也受HIF-1α調(diào)節(jié)，HIF-1α被HCV Core蛋白上調(diào)，并增強肝癌細(xì)胞的遷移性和滲透性[69]。

2.5 宿主因子在HCV-HCC中的作用

2.5.1癌基因突變與HCC目前已確定幾個與肝癌發(fā)生相關(guān)的基因突變[70]。腫瘤抑制基因p53突變導(dǎo)致基因失活或產(chǎn)生顯性陰性型[28]。全基因組測序發(fā)現(xiàn)，編碼Wnt信號通路的β-catenin蛋白的基因具有較高突變率(>30%)[71]。端粒酶反轉(zhuǎn)錄酶(telomerase reverse transcriptase，TERT)基因是HCC中另一頻繁發(fā)生遺傳性改變的基因，通常涉及其啟動子區(qū)，導(dǎo)致其表達(dá)增加[61]。

2.5.2宿主基因單核苷酸多態(tài)性(singlenucleotidepolymorphism，SNP)與HCCSNP與疾病的關(guān)系近年來備受關(guān)注。對與HCV治療和自然清除相關(guān)的λ干擾素成員IFNL3/4的研究發(fā)現(xiàn)，IFNL3 mRNA的3′-非翻譯區(qū)(untranslated region，UTR)多態(tài)性通過影響與HCV感染誘導(dǎo)產(chǎn)生的微小RNA(microRNA，miRNA)結(jié)合而控制其轉(zhuǎn)錄穩(wěn)定性[72-73]。據(jù)報道，IFNL3的多態(tài)性與HCC發(fā)生風(fēng)險升高相關(guān)，特別是在未達(dá)到SVR的患者，但有待進(jìn)一步證實[74]。其他細(xì)胞因子和(或)相關(guān)受體的多態(tài)性也與HCC發(fā)生相關(guān)。如攜帶低單倍型IL-10和TNF-α GG基因型的患者發(fā)生HCC的風(fēng)險更高，IL-23R的罕見突變體與埃及患者中HCV-HCC風(fēng)險降低相關(guān)，與VEGF基因高表達(dá)相關(guān)的多態(tài)性也與HCC相關(guān)[65,75-76]。最近，日本一項大型全基因組關(guān)聯(lián)研究發(fā)現(xiàn)了與HCV-HCC相關(guān)的遺傳變異基因：DEPDC5和人類主要組織相容性復(fù)合體Ⅰ類鏈相關(guān)基因A(major histocompatibility complex class I-related chain A，MICA)[77]。DEPDC5功能未知，MICA可結(jié)合NK細(xì)胞活化性受體凝集素樣同型二聚體(natural killer group 2, member D，NKG2D)，在腫瘤免疫監(jiān)視中介導(dǎo)靶細(xì)胞的毒性。此外，已發(fā)現(xiàn)位于MICA上游的HCP5基因的新型突變體，其對HCC的預(yù)測性更好[78]。在不同種族中進(jìn)一步驗證分析將有助于確認(rèn)這些標(biāo)記的預(yù)測價值。

2.5.3HCV基因序列變異與HCCHCV基因變異也影響HCC發(fā)展。有研究發(fā)現(xiàn)，Core、NS3和NS5A蛋白在HCV 1b及Core在HCV 1a中的多態(tài)性與HCC發(fā)生相關(guān)[79-80]。Harouaka等比較腫瘤與非腫瘤組織HCV序列后，發(fā)現(xiàn)存在不同HCV準(zhǔn)種，提示HCV特定變異株更易誘發(fā)HCC[81]。

2.5.4表觀遺傳學(xué)改變與HCC表觀遺傳調(diào)節(jié)基因的失調(diào)也與HCC發(fā)生相關(guān)，如具有DNA甲基化作用的組蛋白賴氨酸N-甲基轉(zhuǎn)移酶EZH2和重組人蛋白質(zhì)精氨酸甲基轉(zhuǎn)移酶1(protein arginine methyltransferase 1，PRMT1)[82-83]。其中，EZH2通過靶向多種基因在HCC中異常表達(dá)，PRMT1抑制則與HCV介導(dǎo)的蛋白磷酸酶2A催化性C亞基(protein phosphatase 2A catalytic subunit C，PP2Ac)相關(guān)。此外，多種腫瘤抑制基因(包括來自HCV的CDKN2A、GSTP1、RUNX3、APC、SOCS-1和RASSF1A)的高度甲基化也與HCC發(fā)生相關(guān)[84-85]。

2.6 非編碼RNA與HCV-HCC

非編碼RNA泛指一類不編碼蛋白質(zhì)的RNA，根據(jù)長度可劃分為miRNA和長鏈非編碼RNA(long non-coding RNA，lncRNA)等。miRNA和lncRNA的變化均可介導(dǎo)HCC相關(guān)的表觀遺傳改變。

2.6.1miRNA與HCV-HCC已報道多種miRNA可調(diào)節(jié)HCV復(fù)制。其中最具代表性的是miR-122，可與HCV基因組RNA 5′-UTR結(jié)合而增強肝細(xì)胞內(nèi)的病毒復(fù)制[86]。miRNA在HCV-HCC中扮演不同角色，有些抑制增殖、脂質(zhì)代謝和肝細(xì)胞生長的miRNA(如miR-122、miR-27a和miR-181c)被下調(diào)，而其他可調(diào)節(jié)代謝和免疫反應(yīng)的miRNA(如miR-21、miR-221、miR-130a、Let7b、miR-155和miR-200c)被上調(diào)[87]。最近發(fā)現(xiàn)，miR-200c和miR-21的上調(diào)與HCV患者的肝纖維化相關(guān)[87]。

2.6.2lncRNA與HCV-HCClncRNA可調(diào)節(jié)腫瘤抑制基因或癌基因的表達(dá)[88]。HCC相關(guān)lncRNA通過表觀遺傳沉默、剪接調(diào)節(jié)、lncRNA-miRNA相互作用、lncRNA-蛋白相互作用和遺傳變異等多種機制，參與HCC進(jìn)展的多樣化生物過程，如細(xì)胞增殖、凋亡、侵襲、轉(zhuǎn)移和血管發(fā)生等[89-90]。研究發(fā)現(xiàn)，在HCV-HCC的不同發(fā)展階段，lncRNA LINC01419的表達(dá)在早期HCC組織中較發(fā)育不良組織明顯增加，而lncRNA AF070632在晚期HCC組織中比早期HCC組織減少。此外，與非腫瘤性肝組織相比，LINC01419和AK021443的表達(dá)在HCC組織中上調(diào)?；蚬脖磉_(dá)網(wǎng)絡(luò)分析結(jié)果提示，LINC01419和AK021443主要參與細(xì)胞周期進(jìn)展，而AF070632與輔因子結(jié)合、氧化還原和羧酸分解代謝過程相關(guān)[91]。Kamel等報道了兩種lncRNA：lncRNA-尿路上皮癌胚抗原1(urothelial carcinoma associated-1，UCA1)和p53反義轉(zhuǎn)錄本W(wǎng)RAP53(WD repeat containing，antisense to TP53)。他們發(fā)現(xiàn)HCC患者的UCA1和WRAP53血清水平高于慢性HCV感染患者或健康志愿者，腫瘤組織中UCA1和WRAP53的表達(dá)高于相鄰的非腫瘤組織，血清和組織中UCA1與WRAP53的表達(dá)存在顯著相關(guān)性。此外，UCA1和WRAP53的上調(diào)與Child-Pugh評分相關(guān)。Kaplan-Meier分析顯示，UCA1和WRAP53陽性HCC患者具有顯著降低的無復(fù)發(fā)生存期(relapse-free survival，RFS)。Cox多變量分析顯示，WRAP53是一個獨立的RFS預(yù)后因子[92]。這些發(fā)現(xiàn)有助于更好地了解lncRNA在HCV-HCC發(fā)展中的作用。

2.6.3環(huán)狀RNA分子(circularRNA，circRNA)與HCV-HCCcircRNA是一類不具有5′端帽子和3′端poly(A)尾巴并以共價鍵形成環(huán)形結(jié)構(gòu)的非編碼RNA分子。相較于miRNA和lncRNA，circRNA具有更高的穩(wěn)定性和序列保守性[93]。多項研究已報道circRNA與HCC有關(guān)。Shang等發(fā)現(xiàn)hsa_circ_0005075在HCC組織中明顯升高，并與腫瘤大小相關(guān)，在HCC發(fā)展中促進(jìn)細(xì)胞黏附[94]。Xu等發(fā)現(xiàn)ciRS-7在HCC組織中明顯升高，與肝癌微血管侵犯、甲胎蛋白(α fetoprotein，AFP)水平及低齡明顯相關(guān)，且部分影響HCC惡化[95]。Qin等發(fā)現(xiàn)hsa_circ_0001649在HCC組織中明顯降低，其可能在HCC形成及轉(zhuǎn)移過程中起重要作用[96]。circRNA在HCV-HCC發(fā)展中的作用仍需進(jìn)一步闡明。

3 結(jié)語

HCV慢性感染是HCC發(fā)生的重要危險因素，對HCV-HCC發(fā)病機制的研究在世界范圍內(nèi)已引起極大關(guān)注。闡明HCV如何通過直接和間接機制觸發(fā)HCC，鑒別與HCC相關(guān)的基因和表觀遺傳因子，將有助于監(jiān)測慢性肝炎-肝硬化-肝癌的發(fā)展，確認(rèn)高風(fēng)險HCV患者并探索相應(yīng)干預(yù)措施。隨著DAA的不斷上市，HCV被稱為可“治愈”的肝炎病毒。一項前瞻性研究表明，盡管DAA不會增加HCV患者患肝硬化和肝癌的風(fēng)險，但如果患者在DAA治療前已存在未發(fā)現(xiàn)的肝硬化或肝癌，DAA的使用將會加重肝癌惡化且更加難以治療[8]。近期一項HCV-HCC患者接受單純DAA治療的研究顯示，早期HCC的高復(fù)發(fā)率與HCV的清除率一致，提示DAA對腫瘤免疫監(jiān)視可能存在抑制作用[97]。盡管目前HCV蛋白對宿主信號通路調(diào)節(jié)作用的研究已取得可喜成果，但這些結(jié)果多基于體外細(xì)胞模型獲得，未來仍需進(jìn)一步的體內(nèi)研究證實?？傊?，研究HCV-HCC的分子機制，闡明肝臟免疫學(xué)及分子微環(huán)境在HCV-HCC進(jìn)展中的作用，識別肝癌發(fā)生和發(fā)展的預(yù)測指標(biāo)，將為人類戰(zhàn)勝HCV感染奠定基礎(chǔ)。

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. REN Hao, E-mail: hren2013@139.com

MolecularmechanismsofhepatitisCvirus-inducedhepatocellularcarcinoma

GAO Rong, XU Li, XU Jie, REN Hao

DepartmentofMicrobiology,ShanghaiKeyLaboratoryofMedicalBiodefense,TheSecondMilitaryMedicalUniversity,Shanghai200433,China

Hepatitis C virus (HCV) is a leading cause of hepatocellular carcinoma (HCC). HCV-induced hepatocarcinogenesis is a sequential process caused by viral factors and (or) chronic host inflammation status. Host genetic variation is now emerging as an additional element that contributes to one’s risk of developing HCC. Therefore, a comprehensive understanding of the molecular mechanisms for HCV-induced HCC will help to solve this problem.

Hepatitis C virus; Hepatocellular carcinoma; Molecular mechanism

國家自然科學(xué)基金(31370196)

任浩

2017-04-11)