孫凌云，李星逾，孫志為

1.昆明理工大學(xué)醫(yī)學(xué)院,昆明 650500；

2.昆明理工大學(xué)附屬醫(yī)院肝膽外科，昆明 650032

原發(fā)性肝癌的表觀遺傳學(xué)及其治療

孫凌云1,2，李星逾2，孫志為2

1.昆明理工大學(xué)醫(yī)學(xué)院,昆明 650500；

2.昆明理工大學(xué)附屬醫(yī)院肝膽外科，昆明 650032

肝癌是一種嚴(yán)重危害人類健康的惡性疾病，在全世界患癌人群中，肝癌的發(fā)生率排第五，死亡率排第二。原發(fā)性肝癌(Hepatocellular carcinoma,HCC)是最普遍的肝癌組織學(xué)亞型，屬于異質(zhì)性疾病，對(duì)其治療涉及遺傳學(xué)、基因組學(xué)、環(huán)境毒理學(xué)等多個(gè)領(lǐng)域。盡管許多分子靶向治療藥物如索拉菲尼等已經(jīng)進(jìn)入臨床應(yīng)用并證明有效，但細(xì)胞毒性等負(fù)效應(yīng)不容忽視，目前迫切需要新的治療靶點(diǎn)和藥物高效并選擇性的殺傷肝癌細(xì)胞。大量證據(jù)表明，肝臟腫瘤的發(fā)生和發(fā)展與表觀遺傳學(xué)密切相關(guān)，DNA甲基化、組蛋白修飾、miRNA表達(dá)的異常及表觀遺傳相關(guān)基因表達(dá)的異常都是HCC中顯著的表觀遺傳異?，F(xiàn)象。表觀治療藥物可能會(huì)逆轉(zhuǎn)異?；虻谋磉_(dá)，從而使HCC的發(fā)生和發(fā)展得以控制。文章綜述了HCC表觀遺傳學(xué)治療方面的研究進(jìn)展，展望了未來利用類似的療法治療肝癌的潛力。

原發(fā)性肝癌；表觀遺傳學(xué)；DNA甲基化；組蛋白修飾；miRNA；lncRNA

肝癌是一種嚴(yán)重危害人類健康的惡性疾病，在全世界患癌人群中，肝癌的發(fā)生率排第五，死亡率排第二。肝癌在南亞和東南亞尤其流行，確診病例占全球50%以上。此外，全球受影響最嚴(yán)重的5個(gè)地區(qū)還包括非洲中部、西部和南部[1]。原發(fā)性肝癌(Hepatocellular carcinoma,HCC)是最普遍的肝癌組織學(xué)亞型。在過去的10年間，美國(guó)HCC的發(fā)病率已經(jīng)增至3倍，這種增長(zhǎng)與高頻率丙型肝炎病毒(Hepatitis C virus,HCV)感染密切相關(guān)[2,3]?；糎CC的危險(xiǎn)因素主要包括慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染、丙型肝炎病毒感染、過量飲酒、糖尿病、非酒精性脂肪肝以及飲食攝入黃曲霉素[4]等。

1 HCC治療所面臨的挑戰(zhàn)

HCC是一種異質(zhì)性疾病，對(duì)其治療涉及遺傳學(xué)、基因組學(xué)、環(huán)境毒理學(xué)等多個(gè)領(lǐng)域。臨床上面臨的主要挑戰(zhàn)之一是在早期無法檢測(cè)到HCC的發(fā)生，患者經(jīng)常在晚期才被檢查出患病而確診，延誤了早期治療的最佳時(shí)期，從而限制了治療方法的選擇并且導(dǎo)致預(yù)后較差、治療效果不理想[5]。目前，HCC的治療方法主要包括：(1)外科手術(shù)切除腫瘤和肝移植；(2)利用射頻消融術(shù)或冷凍消融術(shù)進(jìn)行最小程度的侵入性外科手術(shù)；(3)直接向肝內(nèi)注射藥物進(jìn)行肝動(dòng)脈栓塞化療[6]。然而，由于存在以下原因，如：缺乏器官捐贈(zèng)者，僅有少部分的患者適合進(jìn)行手術(shù)切除，術(shù)后復(fù)發(fā)率高以及存在諸如肝硬化、HBV和HCV感染的潛在并發(fā)癥[7]等，限制了外科手術(shù)以及肝動(dòng)脈栓塞化療的應(yīng)用。

到目前為止，只有部分基于分子水平的療法在HCC的臨床治療上是可行的。2007年，索拉菲尼獲美國(guó)食品和藥物管理局批準(zhǔn)用于晚期HCC的治療。這種多酪氨酸激酶抑制劑通過干擾血管內(nèi)皮生長(zhǎng)因子信號(hào)通路最終阻斷腫瘤血管的生成。臨床試驗(yàn)表明，該藥使患者中位存活期延長(zhǎng)了3個(gè)月[8]。亞非地區(qū)的一組患者經(jīng)索拉菲尼治療后，中位總生存期從4.2個(gè)月延長(zhǎng)到6.5個(gè)月[9]。進(jìn)行中的臨床試驗(yàn)正在結(jié)合其他藥物測(cè)試索拉菲尼的療效[10]。表皮生長(zhǎng)因子受體、肝細(xì)胞生長(zhǎng)因子及其受體c-Met、血小板生長(zhǎng)因子受體、哺乳動(dòng)物類雷帕霉素靶蛋白均參與了與生長(zhǎng)因子有關(guān)的分子通路[11]，針對(duì)這些靶點(diǎn)的其他藥物也在研究過程中，但這些藥物對(duì)HCC的治療效果都不太穩(wěn)定，或引起嚴(yán)重的不良反應(yīng)。

2 表觀遺傳學(xué)與肝癌的關(guān)系

表觀遺傳學(xué)是研究在不改變DNA序列的前提下，通過某些機(jī)制引起可遺傳的基因表達(dá)或細(xì)胞表型變化的學(xué)科。表觀遺傳學(xué)包括DNA甲基化(DNA methylation)、基因組印記(Genomic imprinting)、母體效應(yīng)(Maternal effects)、基因沉默(Gene silencing)、核仁顯性、休眠轉(zhuǎn)座子激活和RNA編輯(RNA editing)等。表觀遺傳機(jī)制包括基因組DNA的修飾(DNA中胞嘧啶堿基的甲基化)、組蛋白尾部的化學(xué)修飾以及非編碼miRNA的調(diào)控。在細(xì)胞分裂過程中，這些表觀遺傳修飾保守傳遞給子代細(xì)胞從而使“細(xì)胞的記憶”得以維持[12]。

DNA甲基轉(zhuǎn)移酶(DNAmethyltransferases,DNMTs)催化甲基基團(tuán)(CH3)加入到5'端胞嘧啶核苷酸上?；騿?dòng)子區(qū)的CpG島一般處于非甲基化狀態(tài)，當(dāng)其表達(dá)量異常增高時(shí)，會(huì)造成某些基因沉默，這些基因包括DNA修復(fù)基因、抑癌基因、細(xì)胞凋亡基因、細(xì)胞周期調(diào)節(jié)基因等，其中抑癌基因表達(dá)沉默是誘發(fā)腫瘤的重要因素之一。DNA甲基化導(dǎo)致轉(zhuǎn)錄后基因沉默的機(jī)制有兩種：(1)位于CpG島上的甲基化在空間上阻礙轉(zhuǎn)錄因子與它們各自基因啟動(dòng)子的同源結(jié)合位點(diǎn)相結(jié)合[13]；(2)發(fā)生甲基化后，甲基化DNA結(jié)合蛋白可以和該段甲基化DNA相結(jié)合，從而抑制轉(zhuǎn)錄[14]。研究發(fā)現(xiàn)，在許多癌癥中都存在由DNA甲基化介導(dǎo)的基因沉默。正常的DNA甲基化能夠維持基因的穩(wěn)定性，減少基因組中因同源重組引起的染色體缺失、重復(fù)出現(xiàn)等。所以，基因組出現(xiàn)低甲基化狀態(tài)時(shí)也可誘發(fā)腫瘤。癌癥的發(fā)生通常伴隨有基因組整體低甲基化以及抑癌基因啟動(dòng)子的高甲基化[15]。一系列的研究表明，表觀遺傳學(xué)改變與肝癌發(fā)生和進(jìn)展密切相關(guān)[16]。

近期多個(gè)研究報(bào)道了肝癌中啟動(dòng)子甲基化的基因，基因組甲基化圖譜研究證實(shí)：與癌旁正常肝細(xì)胞相比，HCC腫瘤細(xì)胞呈現(xiàn)出特殊的甲基化模式。Hernandez-Vargas等[17]利用基因芯片檢測(cè)了30位患有HBV或HCV感染引起的HCC的患者體內(nèi)1505個(gè)CpG位點(diǎn)，發(fā)現(xiàn)HCC腫瘤細(xì)胞呈現(xiàn)出特殊的DNA甲基化模式，并且DNA甲基化狀態(tài)與主要感染因素和腫瘤發(fā)展進(jìn)程密切相關(guān)。尤其是HCC細(xì)胞中某些基因(如APC、RASSFIA、CDKN2A和FZD7)啟動(dòng)子區(qū)甲基化狀態(tài)與癌旁正常肝臟組織明顯不同，特別是DNMT1啟動(dòng)子甲基化與腫瘤的低分化密切相關(guān)。Song等[18]測(cè)定了27位HCC患者腫瘤組織以及癌旁正常肝臟組織的DNA甲基化水平，結(jié)果發(fā)現(xiàn)啟動(dòng)子區(qū)CpG島DNA甲基化位點(diǎn)顯著富集，這些位點(diǎn)在細(xì)胞生長(zhǎng)、基因表達(dá)、細(xì)胞凋亡以及癌變的信號(hào)通路中發(fā)揮著重要作用，其中BMP4、CDKN2A、GSTP1和 NFATC1基因啟動(dòng)子區(qū)CpG島DNA甲基化水平最高。Shen等[19]對(duì)部分HBV陽性感染的HCC患者血漿進(jìn)行了全基因組甲基化研究，發(fā)現(xiàn)DAB2IP、BMP4、ZFP41、SPDY1和CDKN2A為5大高甲基化基因，而CCL20、ATK3、SCGB1D1、WFDC6和PAX4為5大低甲基化基因。

目前，對(duì)于肝癌組蛋白修飾的研究集中于組蛋白中賴氨酸殘基的乙酰化以及甲基化。組蛋白乙?；ǔＥc轉(zhuǎn)錄激活正相關(guān)，但甲基化可能伴有轉(zhuǎn)錄激活或轉(zhuǎn)錄抑制，這取決于組蛋白修飾的位點(diǎn)。研究發(fā)現(xiàn)，在基因的表達(dá)過程中，組蛋白乙?；腿ヒ阴；g的平衡十分重要，組蛋白去乙酰化和轉(zhuǎn)錄受阻之間的失衡與腫瘤的發(fā)生或抑制直接相關(guān)；目前對(duì)于HCC中的組蛋白甲基化狀態(tài)的評(píng)估僅局限于HCC臨床病理特征的相關(guān)研究，主要運(yùn)用蛋白質(zhì)的半定量檢測(cè)方法如免疫組化或者蛋白質(zhì)印跡法。HCC腫瘤中組蛋白H3的第4位賴氨酸的高甲基化與患者總生存期短以及預(yù)后差密切相關(guān)[20]。另有研究表明，組蛋白H3的第27位賴氨酸的高甲基化導(dǎo)致預(yù)后更差，并且增強(qiáng)了腫瘤的侵襲特征，包括血管生成、腫瘤增大、多樣癌變和腫瘤低分化[21]。肝癌細(xì)胞發(fā)生各種類型的組蛋白修飾異常，其中一個(gè)重要原因是組蛋白修飾酶表達(dá)失調(diào)。這些修飾酶包括組蛋白去乙?；?Histone deacetylases,HDACs)I類亞型HDACl、HDAC2、HDAC3和SIRTl，III類HDAC和組蛋白甲基轉(zhuǎn)移酶(Histone methyltransferases, HMTs)SMYD3、RIZl和EZH2等。組蛋白去乙?；改軌蚯宄械鞍踪嚢彼釟埢阴；鶊F(tuán)，并且在通過染色質(zhì)重塑協(xié)調(diào)細(xì)胞內(nèi)部信號(hào)轉(zhuǎn)導(dǎo)途徑相互作用的過程中扮演著重要角色。所以，通過抑制組蛋白修飾酶表達(dá)失調(diào)來阻斷肝癌發(fā)生有望成為肝癌治療的新途徑。

此外，對(duì)于腫瘤發(fā)生、發(fā)展、侵襲和轉(zhuǎn)移的分子機(jī)制的探索已進(jìn)入一個(gè)新的里程，microRNA (miRNA)與腫瘤關(guān)系的研究是其中的一個(gè)熱點(diǎn)。miRNA是一種能夠調(diào)控基因表達(dá)的短鏈非編碼RNA，短發(fā)夾RNA(Short hairpin RNA,shRNA)可被核酸酶剪切形成miRNA或產(chǎn)生能夠進(jìn)行RNA干擾的siRNA。研究發(fā)現(xiàn)，越來越多的miRNA參與編碼基因的表達(dá)調(diào)控，這些基因參與發(fā)育、細(xì)胞分化、代謝調(diào)控、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞增殖和凋亡等一系列生物學(xué)過程，這些生物學(xué)過程的異常最終導(dǎo)致腫瘤的發(fā)生和發(fā)展。某些miRNA參與了抵抗病毒HCV和PFV-1的抗病毒防御機(jī)制，說明miRNA在病毒感染宿主的過程中發(fā)揮著重要的作用[22,23]。Lagos-Quintana等[24]在miRNA文庫(kù)篩選中，發(fā)現(xiàn)miRNA抑制了HepG2細(xì)胞中HBV病毒的表達(dá)和復(fù)制。miRNA在肝臟中表達(dá)量最高的是miR-122[25]，其在HCC細(xì)胞中普遍下調(diào)[26,27]，因此可以考慮通過恢復(fù)miR-122的表達(dá)實(shí)現(xiàn)其作為抑癌基因[28]的作用。相反，也有報(bào)道稱miR-1的過表達(dá)增強(qiáng)了HBV病毒的復(fù)制，而且HBx蛋白抑制了P53介導(dǎo)的miRNA-148a的表達(dá)，最終導(dǎo)致人造血相關(guān)的PBX相互作用蛋白質(zhì)(Hematopoietic PBX—interacting protein,HPIP)的表達(dá)下降[29]。這些研究均表明，miRNA表達(dá)水平的改變?cè)贖CC發(fā)展過程中具有重要意義。

長(zhǎng)鏈非編碼RNA(Long non-coding RNA,lncRNA)是長(zhǎng)度在200~100000 nt之間的RNA分子，屬于非編碼RNA的亞群，通過不同的機(jī)制從分子水平發(fā)揮調(diào)控功能[30]。越來越多的證據(jù)表明，lncRNA與很多細(xì)胞功能相關(guān)并且在許多腫瘤癌變的過程中發(fā)揮重要作用[31]。已有研究表明，20%的lncRNA與PRC2有關(guān)，lncRNA通過PRC2募集和引導(dǎo)染色質(zhì)修飾復(fù)合物與特定的基因組區(qū)域結(jié)合從而調(diào)控基因轉(zhuǎn)錄[32]。lncRNA類似于轉(zhuǎn)錄活化子/抑制子能夠直接與各種配體相結(jié)合。例如lncRNA TERRA可以直接與人的端粒酶結(jié)合并抑制端粒酶活性[33]。此外，lncRNA也可作為誘餌與miRNA競(jìng)爭(zhēng)結(jié)合位點(diǎn)從而調(diào)節(jié)靶基因的表達(dá)[34]。lncRNA HULC是最先被檢測(cè)到在HCC患者血液中高度上調(diào)的lncRNA[35]，通過下調(diào)抑癌基因p18促使肝癌細(xì)胞大量增殖[36]。研究還發(fā)現(xiàn)，HBx下調(diào)一種被稱為lncRNA-Dreh的lncRNA，這種抑癌因子在體內(nèi)和體外均可抑制HCC細(xì)胞的增殖和代謝[37]。大量HCC患者體內(nèi)的lncRNA MALAT-1[38]和lncRNA HOTAIR[39]高度上調(diào)，特別是 lncRNA HOTAIR常被作為肝臟移植后腫瘤復(fù)發(fā)的預(yù)后診斷標(biāo)記物。HCC患者體內(nèi)高表達(dá)的lncRNA MVIH[40]和lncRNA HEIH[41]分別與血管生成和腫瘤復(fù)發(fā)密切相關(guān)，其中l(wèi)ncRNA HEIH能夠與EZH2結(jié)合，從而下調(diào)抑癌基因p16的表達(dá)[41]。

3 治療肝癌的表觀遺傳學(xué)藥物

傳統(tǒng)的分子靶向治療方法主要致力于抑制失控基因表達(dá)和切斷錯(cuò)誤的信號(hào)通路，相比之下，表觀遺傳學(xué)藥物通過逆轉(zhuǎn)表達(dá)異常基因的甲基化狀態(tài)以及組蛋白修飾狀態(tài)進(jìn)行特異性治療[42]。目前共有4種經(jīng)FDA批準(zhǔn)的表觀遺傳學(xué)藥物，其中包括兩種甲基轉(zhuǎn)移酶(DNMT)抑制劑：5-氮雜胞苷和地西他濱，以及兩種組蛋白去乙?；?HDAC)抑制劑：伏立諾他和丙戊酸(Valporic acid)。這些藥物已經(jīng)成功用于治療血液性腫瘤，特別是治療一種骨髓失去造血功能的血癌：骨髓增生異常綜合征[43]。低劑量的5-氮雜胞苷和地西他濱對(duì)于早期白血病細(xì)胞[44,45]以及從早期管腔型乳腺癌組織[44]中分離得到的細(xì)胞表現(xiàn)出抗腫瘤效應(yīng)。CD34+過高是腫瘤復(fù)發(fā)和產(chǎn)生藥物抗性的起因，然而5-氮雜胞苷和地西他濱能降低白血病細(xì)胞和形成性非黏附性乳腺球群細(xì)胞中CD34+的表達(dá)[46]。針對(duì)HCC中表觀遺傳學(xué)改變的藥物見表1。

3.1 治療HCC的DNA甲基化抑制劑

利用離體細(xì)胞[47]和臨床前期小鼠模型[48]進(jìn)行的研究取得了令人矚目的成果，可為HCC的治療和處理提供新的途徑。Andersen等[48]研究表明，DNMT抑制劑折布拉林(Zebularine)能夠有效抑制異種移植小鼠模型中腫瘤的生長(zhǎng)。然而對(duì)Zebularine產(chǎn)生耐藥性的細(xì)胞株則表現(xiàn)出癌基因的上調(diào)進(jìn)而促進(jìn)了肝癌的發(fā)生發(fā)展，這些發(fā)現(xiàn)表明該藥僅對(duì)部分HCC患者有效。另外，Zebularine也可以抑制癌細(xì)胞擴(kuò)散并誘導(dǎo)HepG2細(xì)胞的凋亡。然而，盡管抗凋亡蛋白BCL-2下調(diào)，但抑癌基因P53和P21的甲基化水平并不會(huì)受到Zebularine的影響，這表明存在某個(gè)單獨(dú)的DNA甲基化調(diào)控通路提高了P53和P21蛋白水平[47]。細(xì)胞水平的研究表明，5-脫氧氮雜胞苷也具有抗腫瘤作用，主要通過抑制端粒酶的活性同時(shí)重新激活P16和c-Myc(原癌基因)的表達(dá)從而產(chǎn)生抗腫瘤效果[49]。

DNA甲基化抑制劑能夠使異常基因恢復(fù)到正常狀態(tài)[50]，適用于表觀遺傳學(xué)治療癌癥[51]，但同時(shí)也面臨著諸多挑戰(zhàn)。相對(duì)來說，DNA甲基化抑制劑只在癌癥早期效果較為顯著，這是因?yàn)镈NA甲基轉(zhuǎn)移酶在惡性腫瘤中的表達(dá)量比良性腫瘤高60%，DNA甲基轉(zhuǎn)移酶的表達(dá)水平越高，DNA甲基化抑制劑的有效劑量也就越高[52]。5-脫氧氮雜胞苷具有細(xì)胞毒性，高濃度的5-脫氧氮雜胞苷會(huì)干擾DNA的合成或引起DNA損傷[53]。此外，5-脫氧氮雜胞苷的穩(wěn)定性較差，極易溶于水，且在胞苷脫氨酶的作用下易脫氨；有限的代謝活性也使其無法作為單一藥劑應(yīng)用于臨床[54]。除了使抑癌基因去甲基化，DNA甲基化抑制劑可能對(duì)正常的甲基化序列如反轉(zhuǎn)錄因子或者其他的轉(zhuǎn)座子也有去甲基化作用[55]。5-脫氧氮雜胞苷使LINE-1元件中的L1-ASP序列發(fā)生去甲基化，該序列位于c-MET基因上，因而最終誘導(dǎo)一種不正常的融合轉(zhuǎn)錄本表達(dá)，該轉(zhuǎn)錄本位于內(nèi)含子LINE-1元件與原癌基因c-MET之間。這種不正常轉(zhuǎn)錄本的表觀活化可能是人類腫瘤細(xì)胞系中藥物介導(dǎo)的低甲基化造成的常見后果，在接受去甲基化治療的患者中也可能存在[55]。此外，5-脫氧氮雜胞苷治療腫瘤還會(huì)產(chǎn)生以下副作用：骨髓抑制、惡心、嘔吐以及腹瀉，這可能限制了其在臨床中特別是對(duì)腫瘤的化學(xué)預(yù)防中的應(yīng)用[56,57]。與5-脫氧氮雜胞苷相比，折布拉林(Zebularine)細(xì)胞毒性低、穩(wěn)定性高，但其效價(jià)低，因此治療時(shí)所需劑量較高[58]。

表1 肝癌治療中針對(duì)表觀遺傳修飾的靶向藥物

3.2 治療HCC的組蛋白去乙?；?HDAC)抑制劑

在臨床前期和臨床階段都曾開展過組蛋白去乙?；?HDAC)抑制劑的效應(yīng)研究。在臨床前期研究階段，伏立諾他(Suberoylanilide hydroxamic acid)使HCC細(xì)胞對(duì)P53乙?；痆59]以及與腫瘤壞死因子相關(guān)的凋亡誘導(dǎo)配體(TNF-related apoptosis-inducing ligand,TRAIL)誘導(dǎo)的細(xì)胞凋亡[60]更加敏感，貝利司他(Belinostat)(一種組蛋白去乙?；敢种苿?同樣能夠抑制HCC細(xì)胞株中細(xì)胞生長(zhǎng)[61]。在一個(gè)多因素的臨床Ⅰ/Ⅱ期試驗(yàn)中，研究人員發(fā)現(xiàn)貝利司他能夠使不可進(jìn)行外科切除手術(shù)的晚期HCC患者病情趨于穩(wěn)定[62]。這一臨床試驗(yàn)的另一個(gè)重要成果是，發(fā)現(xiàn)HR23B可以作為一個(gè)潛在的生物標(biāo)記用于預(yù)測(cè)人體對(duì)于貝利司他的響應(yīng)[62]。伏立諾他與二氫青蒿素(Dihydroartemisinin)聯(lián)合用藥明顯抑制了肝癌移植瘤的生長(zhǎng)[63]。值得注意的是，研究人員發(fā)現(xiàn)HDAC抑制劑能夠誘導(dǎo)細(xì)胞調(diào)亡同時(shí)激活促使腫瘤惡化的基因[64]。這些研究結(jié)果表明，想要進(jìn)一步了解人體對(duì)于表觀遺傳學(xué)藥物響應(yīng)的決定性因素，還需要對(duì)表觀遺傳學(xué)機(jī)制進(jìn)行深入探究。

最新發(fā)現(xiàn)的GSK-1屬于表觀遺傳學(xué)藥物家族，是第一個(gè)化學(xué)合成的抑制劑，以組蛋白去甲基化酶JMJD3/UTX(KDM6A/KDM6B)作為靶點(diǎn)，具備特異性和效應(yīng)性[65]。JMJD3在脂多糖刺激的基礎(chǔ)上經(jīng)巨噬細(xì)胞中專有的核因子κB誘導(dǎo)產(chǎn)生，并在炎癥反應(yīng)和表觀遺傳重編程之間建立重要的聯(lián)系[66]。這些組蛋白去甲基化酶(JMJD3/UTX)催化H3K27中三甲基標(biāo)記物的消除。GSK-1能特異性的抑制脂多糖介導(dǎo)的促炎反應(yīng)，該過程是通過下調(diào)腫瘤壞死因子-α（TNF-α）基因的表達(dá)實(shí)現(xiàn)的，具體的下調(diào)機(jī)制為GSK-1對(duì)TNF-α基因上H3K27的抑制作用以及阻礙轉(zhuǎn)錄過程中RNA聚合酶Ⅱ的募集[65]。由HBV和HCV感染引起的慢性炎癥反應(yīng)，或者肥胖，均會(huì)導(dǎo)致肝損傷進(jìn)而緩慢促進(jìn)HCC的發(fā)生發(fā)展，HCC患者因此產(chǎn)生高水平的促炎細(xì)胞因子TNF-α和白介素(Interleukin,IL)-6[67,68]。由于能夠抑制TNF-α的表達(dá)，GSK-1作為治療HCC的特效藥具有很大潛能。

3.3 基于miRNA治療HCC

不同類型的miRNA在肝臟腫瘤中出現(xiàn)調(diào)控異常[69]，該過程涉及某些細(xì)胞內(nèi)信號(hào)通路[70]。另一方面，有研究已經(jīng)提出利用miRNA作為生物標(biāo)志物[71]和有效治療靶點(diǎn)的可能性。基于miRNA作為治療靶點(diǎn)的藥物包括：(1)抗 miRNA的反義寡核苷酸(Anti-miRNAoligonucleotides,AMOS)[72]；(2)RNA拮抗劑[73]；(3)miRNA類似物[71]；(4)特定鎖核酸(Locked nucleic acid,LNA)修飾的寡核苷酸(LNA介導(dǎo)的miRNA抑制劑)[74]。

在HCC中miRNA具有抑制腫瘤的作用，如miR-26a，作為抗腫瘤試劑是可行的。有報(bào)道稱在人類原發(fā)性肝癌中存在miR-26a表達(dá)水平下降的現(xiàn)象[75]，然而在肝臟原癌基因轉(zhuǎn)基因小鼠模型中通過腺病毒介導(dǎo)的基因治療實(shí)現(xiàn)了miR-26a的系統(tǒng)性修復(fù)，從而抑制了腫瘤細(xì)胞的增殖[76]。miR-26a直接通過靶向下調(diào)周期蛋白D2和E2誘導(dǎo)細(xì)胞周期停滯進(jìn)而產(chǎn)生抗腫瘤的作用。重要的是，這種miRNA在主要器官中的細(xì)胞毒性是最小的。此外，在利用化學(xué)致癌劑二乙基亞硝銨誘導(dǎo)肝癌的小鼠模型中，通過系統(tǒng)性的基因治療實(shí)現(xiàn)了miR-124修復(fù)，顯著降低了肝臟腫瘤的大小[77]，在關(guān)鍵臟器中同樣未檢測(cè)到細(xì)胞毒性。

3.4 基于lncRNA治療HCC

鑒于lncRNAs在癌癥中具有重要的調(diào)控功能，并且大量研究表明lncRNAs在肝臟腫瘤中表達(dá)失調(diào)，以lncRNAs為靶點(diǎn)的藥物為肝癌治療提供了新的機(jī)遇。原則上，lncRNA的靶向治療可以通過以下的方法實(shí)現(xiàn)：(1)siRNA介導(dǎo)的基因沉默；(2)功能缺失。利用小分子或寡核苷酸抑制劑阻止lncRNAs與蛋白質(zhì)(如PRC2)的相互作用；(3)結(jié)構(gòu)干擾。利用小分子或寡核苷酸抑制劑來改變或者模擬lncRNAs的二級(jí)結(jié)構(gòu)從而競(jìng)爭(zhēng)其結(jié)合位點(diǎn)。但是針對(duì)lcnRNAs的藥物研究仍處于起步階段，目前還沒有上市[78]。

研究表明，lncRNA MALAT-1[38]和 lncRNA HOTAIR(HOX基因的反義RNA)[39]在大量的HCC患者體內(nèi)上調(diào)，lncRNA HOTAIR可被作為肝臟移植后腫瘤復(fù)發(fā)的生物標(biāo)志物進(jìn)行預(yù)后。這兩項(xiàng)研究都表明，siRNA介導(dǎo)的 lncRNA MALAT-1和 lncRNA HOTAIR的下調(diào)抑制了細(xì)胞活性和細(xì)胞浸潤(rùn)，促進(jìn)了TNF-α誘導(dǎo)的細(xì)胞凋亡，提高了腫瘤細(xì)胞對(duì)順鉑和多柔比星的化療敏感性[39]。

3.5 具有表觀遺傳特性的天然生物活性化合物

膳食營(yíng)養(yǎng)素和天然植物產(chǎn)品具有抗腫瘤特性，幾年來已應(yīng)用于癌癥治療。然而近年來諸多研究表明，天然生物活性化合物能夠通過表觀遺傳途徑的調(diào)控抑制癌癥發(fā)展[79]。事實(shí)上，最近的數(shù)據(jù)已經(jīng)清楚表明，各種天然化合物對(duì)不同腫瘤細(xì)胞系(例如結(jié)腸、前列腺、乳腺)中的表觀遺傳學(xué)相關(guān)酶和基因(如DNMTs、類HDAC和miRNA)具備特異性調(diào)控功能，因此作為治療性藥物和預(yù)防藥劑具有巨大潛力[80]，諸如綠茶多酚EGCG、水飛薊賓(Silibinin)、喜樹堿(CamptothecinL, CPT) 、 雙 氫 青 蒿 素(Dihydroartemisinin,DHA)等等[81]。

4 表觀遺傳學(xué)藥物與現(xiàn)存治療方式相結(jié)合

在HCC患者體內(nèi)已經(jīng)檢測(cè)到某些HDAC的異常表達(dá)以及HDAC3和HDAC5基因拷貝數(shù)的增加[82]，這就為利用HDAC抑制劑治療HCC提供了理論依據(jù)。在潛伏期肝臟腫瘤模型中，與單獨(dú)用藥相比，泛HDAC抑制劑帕比司他(Panobinostat)和索拉菲尼(Sorafenib)聯(lián)合用藥顯著抑制了腫瘤生長(zhǎng)[83]，并且提高了腫瘤移植小鼠的存活率。具體的分子機(jī)制包括細(xì)胞凋亡的誘導(dǎo)、組蛋白3的乙?；?、BIRC5基因的下調(diào)以及CDH1的上調(diào)。該研究結(jié)果意味著這種聯(lián)合用藥可能對(duì)HCC患者產(chǎn)生良好的治療效果。

表觀遺傳學(xué)藥物除了能在病態(tài)條件下改變異常的表觀遺傳修飾狀態(tài)，還能通過促進(jìn)抗原遞呈提高宿主免疫原性。DNMT抑制劑5-脫氧氮雜胞苷的全身性用藥誘導(dǎo)腫瘤/睪丸抗原(Cancer-testis Antigen, CTA)生成，并且通過提高腫瘤化療敏感性增強(qiáng)了化療的效果[84]。在惡性黑色素瘤的炎癥微環(huán)境中，生黑色素細(xì)胞表現(xiàn)出細(xì)胞可塑性，即通過腫瘤壞死因子α(TNF-α)引起細(xì)胞在已分化型與未分化型狀態(tài)間進(jìn)行轉(zhuǎn)換，細(xì)胞會(huì)逐漸對(duì)化療藥物產(chǎn)生抗性[85]。5-脫氧氮雜胞苷在體內(nèi)的免疫調(diào)節(jié)活動(dòng)暗示人們可以將其運(yùn)用于臨床，為黑色素瘤患者設(shè)計(jì)新的基于CTA的化療方法[86]。由于HCC通常是在慢性HBV或者HCV感染導(dǎo)致的炎癥后發(fā)生，并且已檢測(cè)到在HCC患者體內(nèi)IL-6[87]和TNF-α[68]的水平升高，因此上述設(shè)想可以拓展到HCC的治療中。

5 展 望

同其他類型的腫瘤相似，HCC與多基因突變和表觀遺傳畸變有關(guān)。然而與基因突變有關(guān)的HCC不易被治愈，因此在HCC患者中頻繁出現(xiàn)的表觀遺傳畸變成為新的治療靶點(diǎn)[88]。迄今為止，全基因組的低甲基化、啟動(dòng)子高甲基化、miRNA的異常表達(dá)及其他表觀遺傳基因(例如EZH2)的異常表達(dá)都是HCC中顯著的表觀遺傳異?，F(xiàn)象。這些基因在細(xì)胞凋亡和細(xì)胞周期檢查點(diǎn)處發(fā)揮著重要的作用，然而表觀遺傳學(xué)藥物可能會(huì)逆轉(zhuǎn)異?；虻谋磉_(dá)，從而使HCC的發(fā)生和發(fā)展得以控制。令人擔(dān)憂的是，由于表觀遺傳藥物具有非目標(biāo)效應(yīng)，有相反作用的基因可能被激活，因此還需要進(jìn)一步研究表觀遺傳藥物在多種類型腫瘤中的詳細(xì)作用機(jī)理。目前，多種腫瘤靶向治療藥物已進(jìn)入臨床試驗(yàn)階段，進(jìn)一步的研究應(yīng)該致力于如何利用臨床設(shè)備更好地篩查得到能最大程度受益于指定治療方案的患者群體，如根

據(jù)病程以及疾病背景進(jìn)行篩查，同時(shí)還要確定使用哪種藥物能夠取得最佳療效以及何時(shí)給藥。值得一提的是，有效的生物標(biāo)記能夠?yàn)楹Y查對(duì)藥物產(chǎn)生最優(yōu)反應(yīng)并且復(fù)發(fā)可能性低的患者提供有效指導(dǎo)。此外，以往大多數(shù)表觀遺傳藥物主要用于癌癥晚期，這些藥物是否可以被用作預(yù)防性藥物以及是否可以用于高?；颊叽偈蛊浣邮苓M(jìn)一步的治療仍然值得探究。

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(責(zé)任編委:朱衛(wèi)國(guó))

Progress of epigenetics and its therapeutic application in hepatocellular carcinoma

Lingyun Sun1,2,Xingyu Li2,Zhiwei Sun2

1.Medical College,Kunming University of Science and Technology,Kunming 650500,China;
2.Hepatobiliary Surgery,Affiliated Hospital of Kunming University of Science and Technology,Kunming 650032,China

Liver cancer is a severe harmful disease.It is the fifth most frequently diagnosed cancer and second most frequent cause of cancer deaths worldwide.As the most popular histologic subtype of hepatocellular carcinoma (HCC),primary HCC is a heterogeneous disease whose management requires a multidisciplinary approach combining genetics,genomics and environmental toxicology.Although many molecular targeted therapies such as sorafenib have entered clinical application and proven effective,the cytotoxicity and other negative effects cannot be ignored.There is an urgent need to identify new therapeutic targets and drugs,which can kill HCC cells with high efficiency and specificity.Plenty of evidence suggests that occurrence and development of HCC is closely related with epigenetics. DNA methylation,histone modification,aberrant expression of miRNAs and dysregulated expression of many epigenetic regulatory genes are significantly altered in HCC.Epigenetic therapeutic drugs may reverse abnormal gene expression,thus controlling the occurrence and development of HCC.In this review,we summarize the latest research progresses in epigenetics and its therapeutic application in HCC,and the potential treatments to be used in the future.

hepatocellular carcinoma;epigenetics;DNA methylation;histone modification;miRNA;LncRNA

2014-12-15;

2015-03-24

云南省應(yīng)用基礎(chǔ)研究計(jì)劃項(xiàng)目(編號(hào)：2012WS007)資助

孫凌云，碩士研究生，專業(yè)方向：分子靶向治療肝細(xì)胞癌的臨床及實(shí)驗(yàn)。E-mail:sly84@qq.com

孫志為，教授，研究方向：肝膽外科、微創(chuàng)外科、移植外科、消化道腫瘤。E-mail:2833570686@qq.com

10.16288/j.yczz.14-443

時(shí)間:2015-5-13 13:58:26

URL:http://www.cnki.net/kcms/detail/11.1913.R.20150513.1358.002.html