吳海競，付思祺，李倩文，張慧明，陸前進(jìn),郭 重,2，3

1中南大學(xué)湘雅二醫(yī)院皮膚科 湖南省表觀遺傳重點(diǎn)實驗室，長沙 410011 哈佛大學(xué)布萊根婦女醫(yī)院 2皮膚病理科 3病理科，波士頓，美國 02115

黑色素瘤是一種惡性程度高且生存率低的皮膚腫瘤，發(fā)病速度快且容易轉(zhuǎn)移至腦、肝臟和肺等人體重要器官。目前對于黑色素瘤的臨床診斷主要基于組織病理學(xué)標(biāo)準(zhǔn)，包括腫瘤深度、侵襲水平、是否有潰瘍和淋巴結(jié)轉(zhuǎn)移。但組織病理學(xué)標(biāo)準(zhǔn)無法區(qū)分從良性黑色素痣轉(zhuǎn)變?yōu)楹谏亓龅膩喰停酂o法預(yù)判哪些患者容易發(fā)生轉(zhuǎn)移，因此，目前研究重點(diǎn)為盡快找到一種可用于黑色素瘤早期診斷和預(yù)測轉(zhuǎn)移可能性的新型且有效的生物標(biāo)志物。此外，雖然目前化學(xué)療法和免疫療法發(fā)展迅速，如威羅菲尼/拉菲尼 (BRAF 抑制劑)[1- 4], 納武單抗(PD- 1抗體)[5]，易普利姆瑪(CTLA- 4 抗體)[6]以及派姆單抗[7]等藥物，可延長患者生存期，但大多數(shù)患者最終發(fā)生耐藥，預(yù)后不佳。因此，可預(yù)測藥物反應(yīng)性的生物標(biāo)志物可能實現(xiàn)針對黑色素瘤的個體化和靶向治療，為黑色素瘤的有效控制帶來曙光。

迄今為止，已發(fā)現(xiàn)一系列新的生物標(biāo)志物，其中一些可預(yù)測黑色素瘤轉(zhuǎn)移、藥物反應(yīng)性等，甚至有些可作為治療靶點(diǎn)。本文將從基因組學(xué)到表觀遺傳學(xué)水平，對新發(fā)現(xiàn)的生物標(biāo)志物進(jìn)行全面系統(tǒng)總結(jié)，討論生物標(biāo)志物臨床應(yīng)用的可行性，為改善患者生活質(zhì)量、延長患者生存期等提供潛在的實驗室指標(biāo)和治療靶點(diǎn)。

1 黑色素瘤的基因生物標(biāo)志物

二代測序技術(shù)(next-generation sequencing，NGS)的發(fā)展成功幫助科學(xué)家們發(fā)現(xiàn)疾病新的潛在致病基因。事實上，目前基因分析已投入臨床應(yīng)用。例如，在應(yīng)用抑制BRAF藥物治療之前，醫(yī)生會應(yīng)用基因分析確定腫瘤細(xì)胞的BRAF 基因是否發(fā)生突變。一項來自TCGA的研究從331例患者333份黑色素瘤標(biāo)本中提取了DNA、RNA和蛋白質(zhì)，并繪制出癌癥基因圖譜，構(gòu)建了不同突變基因(BRAF、NRAS、KIT、GNAQ/GNA11、TP53/CDKN2A和NF1)的分類體系[8]，可指導(dǎo)治療策略的制定(表1)。從全基因組測序(whole-genome sequencing，WGS)和全外顯子測序(whole-exsome sequencing，WES)的研究中發(fā)現(xiàn)，黑色素瘤患者存在高突變率及高紫外線暴露率的特征，而紫外線照射正是黑色素瘤的高危風(fēng)險因素[8- 10]。通過Bonfferoni統(tǒng)計學(xué)方法檢驗(P<0.05)，癌癥基因圖譜研究新發(fā)現(xiàn)了13個黑色素瘤致病基因，分別為BRAF、 NRAS、TP53、NF1、CDKN2A、ARID2、 PTEN、PPP6C、RAC1、 IDH1、DDX3X、MAP2K1和RB1[8]，然而，一些經(jīng)典的導(dǎo)致其他部位癌癥的致病基因如PREX2、GRIN2A、ERBB4、ADAMTS18、BCL2L12、SOX10、MITF和KIT, 卻未在此癌癥基因圖譜中出現(xiàn)，可能由不同的統(tǒng)計學(xué)方法差異所導(dǎo)致[11]。

基因除了可作為生物標(biāo)志物用于腫瘤診斷，基因突變還可預(yù)測其轉(zhuǎn)移可能性及對靶向藥物治療抵抗的概率。例如，NMDAR2 和 EGFR4 突變、MET 和MITF表達(dá)增加以及PTEN表達(dá)丟失預(yù)示轉(zhuǎn)移可能性大，而MITF 和BRAF表達(dá)增加及PTEN表達(dá)丟失則預(yù)示可能對靶向治療抵抗。NRAS突變是目前發(fā)現(xiàn)的唯一一個可預(yù)測免疫治療敏感性的基因標(biāo)志物[23]。這些證據(jù)表明基因分析對監(jiān)控疾病進(jìn)展和指導(dǎo)個體化治療具有重要作用。

表 1 黑色素瘤靶向治療的基因生物標(biāo)志物

除了這些研究較多的基因外，其他候選基因也有可能作為生物標(biāo)志物。如在既往研究中，有學(xué)者試圖將整合素信號通路基因的單核苷酸多態(tài)性(single nucleotide polymorphisms，SNPs)與皮膚黑色素瘤存活率相關(guān)聯(lián)。德克薩斯大學(xué)M.D.安德森癌癥中心的全基因組相關(guān)性研究發(fā)現(xiàn)，整合素基因通路中3個獨(dú)立的SNPs：DOCK1 rs11018104 T>A、 rs35748949 C>T和PAK2 rs1718404 C>T有望成為預(yù)測黑色素瘤預(yù)后的生物標(biāo)志物[24]；在肢端雀斑樣黑色素瘤中發(fā)現(xiàn)TERT變異，TERT抑制物可能成為潛在治療策略，使得TERT變異成為制定黑色素瘤個體化治療策略的潛在生物標(biāo)志物[25]；從33例葡萄膜黑色素瘤患者中的WGS研究發(fā)現(xiàn)，葡萄膜黑色素瘤的可能致病基因為GNAQ、GNA11、BAP1、 EIF1AX和 SF3B1, 并發(fā)現(xiàn)了體細(xì)胞突變中的其他基因如TP53BP1、CSMD1、TTC28DLK2及KTN1[26]，但仍需進(jìn)一步擴(kuò)大樣本量確定這些基因突變是否與葡萄膜黑色素瘤的發(fā)生發(fā)展具有緊密聯(lián)系。

2 黑色素瘤的表觀遺傳學(xué)生物標(biāo)志物

表觀遺傳學(xué)是指在基因表達(dá)和功能中不涉及DNA核苷酸原始序列改變的潛在遺傳學(xué)改變，可最終決定基因表達(dá)或沉默，因此在細(xì)胞分化、生長、發(fā)育、老化和免疫反應(yīng)等生命過程中發(fā)揮了關(guān)鍵作用。表觀遺傳學(xué)為環(huán)境因素如何決定個體表型差異提供了除遺傳學(xué)以外的解釋，同時也為一些疾病如黑色素瘤除遺傳易感性以外的發(fā)病機(jī)制提供了證據(jù)。隨著表觀遺傳學(xué)時代的到來，已發(fā)現(xiàn)越來越多表觀遺傳學(xué)生物標(biāo)志物涉及DNA甲基化/羥甲基化、組蛋白修飾和非編碼RNA[微小RNA (micro-RNA, miRNA)和長鏈非編碼RNA(long non-coding RNA，lncRNA)]。對比靶向治療的基因生物標(biāo)志物，表觀遺傳學(xué)治療具有可控、可逆等優(yōu)點(diǎn)。

2.1 DNA甲基化/羥甲基化

在許多真核物種中，DNA甲基化是相對穩(wěn)定并可遺傳的表觀遺傳學(xué)標(biāo)志物，其是指甲基添加到腺嘌呤或胞嘧啶中嘧啶環(huán)的第5個位置，使得胞嘧啶成為甲基胞嘧啶。DNA高甲基化代表基因表達(dá)受到抑制，因此涉及很多生命過程，如細(xì)胞分化與增殖。DNA甲基化由甲基化轉(zhuǎn)移酶如DNMT1、 DNMT3a 和DNMT3b等介導(dǎo)。每一種甲基化轉(zhuǎn)移酶的功能有所差異，如DNMT1在細(xì)胞復(fù)制過程中維持甲基化狀態(tài)，而DNMT3a和DNMT3b常常誘導(dǎo)甲基化的初始化過程。相反，DNA去甲基化是甲基化修飾丟失的過程。DNA甲基化消除主要涉及兩種途徑：一是DNA被動去甲基化, 即在 DNA 復(fù)制時，DNA 甲基化模式的維持受到干擾，未保留原有甲基化模式，隨著復(fù)制的進(jìn)行，甲基化的CpG被“稀釋”，導(dǎo)致 DNA 去甲基化；二是DNA主動去甲基化，這一過程不依賴于DNA復(fù)制，而受酶的催化, 使5-甲基化胞嘧啶(5-methylated cytosine, 5mC)轉(zhuǎn)化為未甲基化的胞嘧啶。DNA羥甲基化介導(dǎo)的主動去甲基化途徑主要包括兩種:(1)氧化去甲基化途徑: 5-羥甲基化胞嘧啶(5-hydroxymethylated cytosine, 5-hmC)先被 TET 蛋白催化轉(zhuǎn)變?yōu)?5-氟胞嘧啶(5-formylcytosine, 5fC)，后者繼續(xù)在TET蛋白的催化作用下轉(zhuǎn)變?yōu)?5-羧基胞嘧啶(5-carboxylcytosine, 5caC)，然后被胸腺嘧啶DNA糖基化酶(thymine DNA glycosylase,TDG)脫羧還原為胞嘧啶[27]；(2)另外一條途徑為5fC直接被TDG脫羧還原為胞嘧啶。這個過程中，5mC氧化成5-hmC是反應(yīng)的第一步也是關(guān)鍵一步，這一步由TET家族(TET1、TET2、TET3)加雙氧酶調(diào)節(jié)[28]。5-hmC是活性DNA去甲基化最豐富的中間媒介物，在正常發(fā)育和癌癥發(fā)生中發(fā)揮正轉(zhuǎn)錄調(diào)控作用[29]，其含量直接與人體各個組織器官的分化水平相關(guān)[30]。

在早期研究中，腫瘤發(fā)生發(fā)展過程中發(fā)現(xiàn)全基因組DNA低甲基化[31]。有研究認(rèn)為DNA低甲基化可促進(jìn)早期腫瘤細(xì)胞增殖和后期轉(zhuǎn)移，使其具有生存優(yōu)勢[32]，且在著絲粒序列和重復(fù)序列方面與染色體不穩(wěn)定相關(guān)[33]。另一方面，在啟動子區(qū)CpG島的DNA甲基化被認(rèn)為通過沉默抑癌基因促進(jìn)腫瘤發(fā)生。許多高甲基化的抑癌基因，參與生物學(xué)過程，包括細(xì)胞周期調(diào)控、DNA修復(fù)、細(xì)胞信號傳導(dǎo)、基因轉(zhuǎn)錄和細(xì)胞凋亡，如已報道的其在黑色素瘤中作為生物標(biāo)志物的潛在可能性[ 34]。

除CDKN2A, RAR-b2, RASSF1A和IDH1[35]等已被其他學(xué)者廣泛研究的基因外，黑色素瘤中還發(fā)現(xiàn)其他高甲基化的基因。如在幾項癌癥研究中發(fā)現(xiàn)LINE- 1是全基因組甲基化的臨床試驗替代標(biāo)志物，并在巴西的黑色素瘤患者中發(fā)現(xiàn)LINE- 1高甲基化，可能成為皮膚黑色素瘤的生物標(biāo)志物[36]。在不同腫瘤類型中還發(fā)現(xiàn)LINE- 1甲基化狀態(tài)與癌癥風(fēng)險因素相關(guān)[37]。此外， Claudin11被認(rèn)為是潛在的診斷黑色素瘤的表觀遺傳學(xué)生物標(biāo)志物[38]，Claudin基因家族包含27個成員，其編碼細(xì)胞間緊密連接的膜蛋白。腫瘤轉(zhuǎn)移灶的位置與甲基化頻率顯著相關(guān)，表明原發(fā)性黑色素瘤的甲基化水平可能有助于判斷黑色素瘤轉(zhuǎn)移能力的差異。因此，研究Claudin11失活分析功能改變很有意義。此外，MGMT編碼一種修復(fù)蛋白，鳥嘌呤殘基的O6位置去除烷基，其啟動子區(qū)甲基化狀態(tài)已被提議作為膠質(zhì)瘤[ 39 ]、大腸癌[ 40 ]和黑色素瘤[ 41 ]的生物標(biāo)志物。在黑色素瘤患者的腫瘤和血清中發(fā)現(xiàn)MGMT基因的表觀遺傳沉默[42]，提示其在腫瘤發(fā)生發(fā)展中的重要作用。MITF是另一個DNA高甲基化基因，一種控制細(xì)胞周期和黑色素生成的轉(zhuǎn)錄因子[43]。在不止一例腫瘤皮損和黑色素瘤患者外周血中發(fā)現(xiàn)MITF啟動子區(qū)高甲基化。有趣的是，不同黑色素瘤標(biāo)本和腫瘤[44]中MITF表達(dá)存在變化，其高表達(dá)代表高度增殖和分化，而相對低表達(dá)可能表明侵襲能力增強(qiáng)[45]，提示MITF基因啟動子高甲基化水平可能與疾病嚴(yán)重程度有關(guān)(表2)。當(dāng)然，仍需更多研究進(jìn)一步明確高甲基化基因在黑色素瘤中的作用，深入探索利用這些甲基化生物標(biāo)志物和治療靶點(diǎn)來治療黑色素瘤。

事實上，運(yùn)用定量甲基化特異性PCR檢測黑色素瘤患者外周血中腫瘤相關(guān)基因的甲基化水平已在臨床應(yīng)用。例如，與對生物化學(xué)療法不敏感的腫瘤相比，循環(huán)中較少甲基化的RASSF1A與生物化學(xué)療法的敏感性相關(guān)，RASSF1A的甲基化水平與總體生存率相關(guān)[68]；黑色素瘤基因組整體表現(xiàn)為低甲基化，更重要的是，5-hmC缺失可作為生物標(biāo)志物來區(qū)分黑色素瘤與生理性黑色素細(xì)胞及良性增殖[69]，且5-hmC缺失與黑色素瘤預(yù)后差具有很強(qiáng)相關(guān)性，預(yù)示5-hmC水平可作為潛在的預(yù)測預(yù)后的生物標(biāo)志物，隨后幾年中的其他研究也進(jìn)一步證實了該發(fā)現(xiàn)[70- 74]；雖然在黑色素瘤中發(fā)現(xiàn)IDH2和TET蛋白水平降低，然而這種改變的上游和下游機(jī)制尚不清楚，TET蛋白已用于癌癥臨床前研究[75]，如沉默TET2和TET3在上皮細(xì)胞向間質(zhì)細(xì)胞轉(zhuǎn)變過程和黑色素瘤轉(zhuǎn)移中發(fā)揮重要作用[76]。

2.2 組蛋白修飾

表 2 黑色素瘤中高甲基化的基因

組蛋白修飾是調(diào)控基因表達(dá)的另一重要表觀遺傳機(jī)制。DNA被包裝成細(xì)胞核作為染色質(zhì)，核小體是染色質(zhì)的基本亞單位。每個核小體由146個堿基對的DNA形成兩圈纏繞在組蛋白核心及兩對H2A、H2B、H3和H4上。組蛋白修飾表現(xiàn)為從核小體凸出的小蛋白尾巴被修飾，包括甲基化、乙酰化和泛素化。每個修飾均有其獨(dú)特的功能，例如組蛋白H3K9乙?；鰪?qiáng)轉(zhuǎn)錄，而甲基化則抑制該過程。在這些修飾中，乙?；兔撘阴Ｗ饔玫玫搅松钊胙芯?，分別由組蛋白乙?；D(zhuǎn)移酶(histone acetyltransferase, HAT)和組蛋白脫乙?；?histone deacetylase，HDAC)催化。HAT將乙酰基轉(zhuǎn)移至賴氨酸，導(dǎo)致基因活化；HDAC去除乙酰基，導(dǎo)致基因沉默。與乙?；煌?，組蛋白甲基化發(fā)生在精氨酸和賴氨酸殘基并受組蛋白甲基轉(zhuǎn)移酶(histone methyltransferase，HMT)和其他酶的調(diào)節(jié)，且甲基化的影響受修飾后殘基位置和甲基基團(tuán)數(shù)目的調(diào)節(jié)，如H3K4me3增加基因表達(dá)，而H3K9me3和H3K27me3則導(dǎo)致基因表達(dá)下降。

組蛋白的改變與黑色素瘤免疫逃逸具有一定關(guān)系。研究發(fā)現(xiàn)組蛋白修飾蛋白和組蛋白修飾酶的異常表達(dá)與黑色素瘤的異常增殖相關(guān)[77]。例如，組蛋白低乙?；驯蛔C明可減少黑色素瘤PI3K/Akt信號通路腫瘤抑制基因的表達(dá)[78]，異常的組蛋白乙?；{(diào)控腫瘤細(xì)胞和腫瘤浸潤免疫細(xì)胞中凋亡途徑TRAIL/Apo2L[79]和Bcl- 2家族成員(Bim、Bax和Bak)[80]的表達(dá)，均提示組蛋白修飾可作為生物標(biāo)志物預(yù)測免疫療法的反應(yīng)或作為HDAC抑制劑的治療靶點(diǎn)。

除了總體組蛋白修飾狀態(tài)外，組蛋白修飾酶也與黑色素瘤的發(fā)展相關(guān)，并有潛力成為生物標(biāo)志物和治療靶點(diǎn)。例如，EZH2是多梳蛋白抑制復(fù)合體上的H3K27甲基轉(zhuǎn)移酶催化亞單位。EZH2的表達(dá)與黑色素瘤中高增殖率和高侵襲性的腫瘤亞型相關(guān)，其通過催化H3K27的轉(zhuǎn)錄從而抑制甲基化[81]。此外， 48%的EZH2基因高表達(dá)患者具有5年總生存期，而71%的EZH2基因低表達(dá)患者具有5年總生存期[81]，說明EZH2基因可預(yù)測黑色素瘤患者的生存期。此外，通過CDKN1A基因乙?；腿ヒ阴；珽ZH2耗盡與黑色素瘤細(xì)胞促凋亡作用相關(guān)[82]?；诤谏亓龈甙l(fā)和易轉(zhuǎn)移的特性，EZH2抑制劑可作為其治療的評估方法[83]。最近一項研究中一種新的定量質(zhì)譜分析確定了伴隨EZH2高表達(dá)，出現(xiàn)組蛋白翻譯后修飾H3K27me3。人類晚期黑色素瘤組織中已發(fā)現(xiàn)EZH2介導(dǎo)的RUNX3基因和腫瘤抑制基因E-cadherin的沉默[84]。另一個例子是SETDB1，在黑色素瘤斑馬魚模型中發(fā)現(xiàn)甲基化組蛋白H3賴氨酸9，可加速黑色素瘤的形成[85]，半數(shù)以上的黑色素瘤患者樣本中檢測出SETDB1的過表達(dá)[86]及其表達(dá)與抑制腫瘤抑制基因p16 的表達(dá)相關(guān)[87]。

2.3 微小RNA

miRNA是21～25個堿基對的非編碼RNA，其通過結(jié)合靶基因信使RNA3’未翻譯區(qū)，使信使RNA裂解，抑制翻譯或使翻譯受阻。研究表明黑色素瘤不同類型細(xì)胞和組織中miRNA表達(dá)異常，因而可能輔助診斷、判斷預(yù)后，甚至作為潛在的治療靶點(diǎn)。與其他標(biāo)志物不同，細(xì)胞死亡裂解后會釋放miRNA進(jìn)入循環(huán)，腫瘤細(xì)胞也可通過外泌體釋放進(jìn)入循環(huán)。

越來越多的證據(jù)表明，miRNA通過調(diào)節(jié)重要的通路或基因發(fā)揮癌基因或抑癌基因作用，這種通路或基因涉及細(xì)胞增殖、凋亡、遷移和侵襲，藥物抵抗和血管生成，如miRNA- 29c和miRNA- 324- 3p在轉(zhuǎn)移性黑色素瘤中不表達(dá)，但具有預(yù)測黑色素瘤轉(zhuǎn)移的作用。事實上，已發(fā)現(xiàn)許多miRNA在黑色素瘤的病理生理過程中發(fā)揮重要作用，如miRNA- 15b、 miRNA- 99a、 miRNA- 137、 miRNA- 148、 miRNA- 149、miRNA- 193b、 miRNA- 211、 miRNA- 221 和miRNA- 506- 514參與細(xì)胞增殖和生長,miRNA- 18b、 miRNA- 26a、 miRNA- 34a、miRNA- 34b/c、miRNA- 137、 miRNA- 203 和 miRNA- 205參與細(xì)胞凋亡，miRNA- 214、miRNA30b/30d、miRNA- 182、 let- 7a、miRNA- 126、miRNA- 145、miRNA- 137、miRNA- 18b、 miRNA- 34a/c、 miRNA- 211、miRNA- 9和miRNA- 31參與細(xì)胞侵襲和轉(zhuǎn)移，miRNA-1908、 miRNA- 199a- 5p 和 miRNA- 199a- 3參與血管生成，miRNA- 200c則參與藥物抗性。

黑色素瘤中研究較多并具有診斷和治療潛能的miRNA包括miRNA- 21、miRNA- 125b、miRNA- 155、miRNA- 205、miRNA- 211等。miRNA- 21在黑色素瘤中表達(dá)增加，通過靶向調(diào)節(jié)TIMP3、PDCD4、BCL- 2和PTEN，在黑色素瘤的發(fā)生發(fā)展中發(fā)揮重要作用[88- 89]；miRNA- 21升高預(yù)示患者5年非進(jìn)展生存期和總生存期下降，故miRNA- 21可幫助判斷預(yù)后[88]。miRNA- 125b在黑色素瘤中表達(dá)下降，通過靶向c-jun、MLK3和MKK7抑制腫瘤細(xì)胞增殖、周期、凋亡和遷移發(fā)揮抑癌基因作用[89]，此外還參與了威羅非尼耐藥[90]。miRNA- 155在黑色素瘤組織中表達(dá)增加，靶向調(diào)節(jié)SK1，促進(jìn)凋亡抑制增殖[91]。而miR- 205和miR- 211在黑色素瘤組織中表達(dá)下降，分別靶向調(diào)節(jié)E-cadherin[92]和NFAT3[93]促進(jìn)腫瘤侵襲。這些miRNA可能作為預(yù)測黑色素瘤分期及進(jìn)展的生物標(biāo)志物。

2.4 長鏈非編碼RNA

lncRNA是長度大于200個堿基對的非編碼RNA。目前，僅有少數(shù)lncRNA進(jìn)行了功能鑒定。根據(jù)其編碼蛋白基因組的接近程度，lncNA分為正義、反義、內(nèi)含子、基因間和雙向5大類。與miRNA不同，lncRNA可正向或負(fù)向調(diào)節(jié)基因表達(dá)，并在lncRNA與RNA、lncRNA與蛋白、lncRNA與染色質(zhì)間形成許多功能。lncRNA是目前的研究熱點(diǎn)，因為大量研究證據(jù)表明通過改變lncRNA的原始結(jié)構(gòu)、繼發(fā)結(jié)構(gòu)和表達(dá)水平，將導(dǎo)致從神經(jīng)退化到癌癥的一系列疾病。雖然目前尚無直接證據(jù)表明lncRNA是黑色素瘤的生物標(biāo)志物，但越來越多的研究表明某些lncRNA的異常表達(dá)與黑色素瘤患者的生存、侵襲和轉(zhuǎn)移息息相關(guān)[94]。例如BANCR與黑色素瘤的生存期相關(guān)，其在原發(fā)和轉(zhuǎn)移黑色素瘤患者中高表達(dá)，并隨腫瘤分級增加而表達(dá)增加，表明在疾病發(fā)展過程中發(fā)揮腫瘤基因功能[95- 96]，更重要的是，BANCR高表達(dá)與低生存期相關(guān)，表明BANCR是預(yù)后差的生物標(biāo)志物[97]；SPRY4-IT1是移植MAPK通路中的腫瘤抑制因子，其在局部黑色素瘤，遠(yuǎn)處轉(zhuǎn)移灶淋巴結(jié)轉(zhuǎn)移的黑色素瘤中表達(dá)增加，預(yù)示了其在黑色素瘤分期和早期診斷的潛在功能[98]；此外，HOTAIR[99]、 CASC15[100]、 MALAT- 1和 UCA1[101]在黑色素瘤中高表達(dá)，可能預(yù)示著黑色素瘤的侵襲性和轉(zhuǎn)移性。但lncRNA是否可成為黑色素瘤診斷和預(yù)后的生物標(biāo)志物，尚需進(jìn)一步研究證實。

3 展望

NGS的普及和表觀遺傳時代的到來，為探索黑色素瘤生物標(biāo)志物提供了強(qiáng)有力的工具。隨著癌基因組學(xué)時代的到來，加之各種先進(jìn)技術(shù)和計算機(jī)模型用于大數(shù)據(jù)分析，聯(lián)合基因組學(xué)、表觀遺傳組學(xué)、蛋白質(zhì)組學(xué)的生物標(biāo)志物可能輔助黑色素瘤和其他良性腫瘤相鑒別。黑色素瘤容易轉(zhuǎn)移，是致死的重要原因，因此發(fā)現(xiàn)器官特異性的生物標(biāo)志物迫在眉睫。此外，血液中的生物標(biāo)志物能幫助早期發(fā)現(xiàn)黑色素瘤、精準(zhǔn)監(jiān)測疾病進(jìn)展和預(yù)測治療效果，從而讓患者受益。因此，開發(fā)新的可用于早期診斷、預(yù)測藥物敏感性、分析轉(zhuǎn)移可能性和判斷預(yù)后的標(biāo)志物，將為黑色素瘤乃至其他惡性腫瘤的治療和預(yù)后提供實驗室指導(dǎo)。

[1] Alcala AM, Flaherty KT. BRAF inhibitors for the treatment of metastatic melanoma: clinical trials and mechanisms of resistance[J]. Clin Cancer Res, 2012, 18: 33- 39.

[2] McArthur GA, Chapman PB, Robert C, et al. Safety and efficacy of vemurafenib in BRAF(V600E) and BRAF(V600K) mutation-positive melanoma (BRIM- 3): extended follow-up of a phase 3, randomised, open-label study[J]. Lancet Oncol, 2014, 15: 323- 332.

[3] Queirolo P, Spagnolo F. BRAF plus MEK-targeted drugs: a new standard of treatment for BRAF-mutant advanced melanoma[J]. Cancer Metastasis Rev, 2017,36:35- 42.

[4] Desvignes C, Abirached H, Templier C, et al. BRAF inhibitor discontinuation and rechallenge in advanced mela-noma patients with a complete initial treatment response[J]. Melanoma Res,2017,27:281- 287.

[5] Beaver JA, Theoret MR, Mushti S, et al. FDA approval of nivolumab for the first-lne treatment of patients with BRAFV600 wild-type unresectable or metastatic melanoma[J]. Clin Cancer Res, 2017, 23:3479- 3483.

[6] Meerveld-Eggink A, Rozeman EA, Lalezari F, et al. Short-term CTLA- 4 blockade directly followed by PD- 1 blockade in advanced melanoma patients - a single center experience[J]. Ann Oncol, 2017, 28:862- 867.

[7] Chuk MK, Chang JT, Theoret MR, et al. FDA approval summary: accelerated approval of pembrolizumab for second-line treatment of metastatic melanoma[J]. Clin Cancer Res, 2017,23:5666- 5670.

[8] Cancer Genome Atlas Network. Genomic classification of cutaneous melanoma[J]. Cell,2015, 161: 1681- 1696.

[9] Krauthammer M, Kong Y, Ha BH, et al. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma[J]. Nat Genet,2012, 44: 1006- 1014.

[10] Lawrence MS, Stojanov P, Polak P, et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes[J]. Nature, 2013, 499: 214- 218.

[11] Zhang T, Dutton-Regester K, Brown KM, et al. The genomic landscape of cutaneous melanoma[J]. Pigment Cell Melanoma Res,2016,29: 266- 283.

[12] Poulikakos PI, Persaud Y, Janakiraman M, et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E) [J]. Nature,2011,480: 387- 390.

[13] Flaherty KT, Robert C, Hersey P, et al. Improved survival with MEK inhibition in BRAF-mutated melanoma[J]. N Engl J Med, 2012, 367: 107- 114.

[14] Long GV, Weber JS, Infante JR, et al. Overall survival and durable responses in patients with BRAF V600-mutant metastatic melanoma receiving dabrafenib combined with trametinib[J]. J Clin Oncol,2016,34: 871- 878.

[15] Robert C, Karaszewska B, Schachter J, et al. Improved overall survival in melanoma with combined dabrafenib and trametinib[J]. N Engl J Med, 2015, 372: 30- 39.

[16] Bahadoran P, Allegra M, Le Duff F, et al. Major clinical response to a BRAF inhibitor in a patient with a BRAF L597R-mutated melanoma[J]. J Clin Oncol,2013, 31: e324- e326.

[17] Marconcini R, Galli L, Antonuzzo A, et al. Metastatic BRAF K601E-mutated melanoma reaches complete response to MEK inhibitor trametinib administered for over 36 months[J]. Exp Hematol Oncol, 2017, 6: 6.

[18] Ascierto PA, Schadendorf D, Berking C, et al. MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study[J]. Lancet Oncol,2013, 14: 249- 256.

[19] Krauthammer M, Kong Y, Bacchiocchi A, et al. Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas[J]. Nat Genet, 2015,47: 996- 1002.

[20] Young RJ, Waldeck K, Martin C, et al. Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines[J]. Pigment Cell Melanoma Res, 2014, 27: 590- 600.

[21] Diller ML, Kudchadkar RR, Delman KA, et al. Complete response to high-dose IL- 2 and enhanced IFNgamma+Th17: TREG ratio in a melanoma patient[J]. Melanoma Res, 2016,26: 535- 539.

[22] Timar J, Vizkeleti L, Doma V, et al. Genetic progression of malignant melanoma[J]. Cancer Metastasis Rev, 2016, 35: 93- 107.

[23] Li H, Wang Y, Liu H, et al. Genetic variants in the integrin signaling pathway genes predict cutaneous melanoma survival[J]. Int J Cancer, 2017,140: 1270- 1279.

[24] Liang WS, Hendricks W, Kiefer J, et al. Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma[J]. Genome Res,2017, 27: 524- 532.

[25] Royer-Bertrand B, Torsello M, Rimoldi D, et al. Comprehensive genetic landscape of uveal melanoma by whole-genome sequen-cing[J]. Am J Hum Genet, 2016,99: 1190- 1198.

[26] Kohli RM, Zhang Y. TET enzymes, TDG and the dynamics of DNA demethylation[J]. Nature,2013, 502: 472- 479.

[27] Tahiliani M, Koh KP, Shen Y, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET[J]. Science,2009, 324: 930- 935.

[28] Cortellino S, Xu J, Sannai M, et al. Thymine DNA glycosylase is essential for active DNA demethylation by linked deamina-tion-base excision repair[J]. Cell, 2011, 146: 67- 79.

[29] Haffner MC, Chaux A, Meeker AK, et al. Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers[J]. Oncotarget, 2011, 2: 627- 637.

[30] Kim YI, Giuliano A, Hatch KD, et al. Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma[J]. Cancer, 1994, 74: 893- 899.

[31] Lee JJ, Murphy GF, Lian CG. Melanoma epigenetics: novel mechanisms, markers, and medicines[J]. Lab Invest,2014,94: 822- 838.

[32] Karpf AR, Matsui S. Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells[J]. Cancer Res, 2005,65: 8635- 8639.

[33] Toyota M, Ahuja N, Ohe-Toyota M, et al. CpG island methylator phenotype in colorectal cancer[J]. Proc Natl Acad Sci USA,1999,96: 8681- 8686.

[34] Sarkar D, Leung EY, Baguley BC, et al. Epigenetic regulation in human melanoma: past and future[J]. Epigenetics, 2015, 10: 103- 121.

[35] De Araujo ES, Kashiwabara AY, Achatz MI, et al. LINE- 1 hypermethylation in peripheral blood of cutaneous melanoma patients is associated with metastasis[J]. Melanoma Res, 2015, 25: 173- 177.

[36] Di JZ, Han XD, Gu WY, et al. Association of hypomethylation of LINE- 1 repetitive element in blood leukocyte DNA with an increased risk of hepatocellular carcinoma[J]. J Zhejiang Univ Sci B,2011,12: 805- 811.

[37] Walesch SK, Richter AM, Helmbold P, et al. Claudin11 promoter hypermethylation is frequent in malignant melanoma of the skin, but uncommon in nevus Cell[J]. Nevi Cancers,2015,7: 1233- 1243.

[38] Cankovic M, Nikiforova MN, Snuderl M, et al. The role of MGMT testing in clinical practice: a report of the association for molecular pathology[J]. J Mol Diagn,2013,15: 539- 555.

[39] Inno A, Fanetti G, Di Bartolomeo M, et al. Role of MGMT as biomarker in colorectal cancer[J]. World J Clin Cases,2014,2: 835- 839.

[40] de Araujo ES, Pramio DT, Kashiwabara AY, et al. DNA methylation levels of melanoma risk genes are associated with clinical characteristics of melanoma patients[J]. Biomed Res Int, 2015, 2015: 376423.

[41] Cheli Y, Ohanna M, Ballotti R, et al. Fifteen-year quest for microphthalmia-associated transcription factor target genes[J]. Pigment Cell Melanoma Res,2010,23: 27- 40.

[42] Ennen M, Keime C, Kobi D, et al. Single-cell gene expression signatures reveal melanoma cell heterogeneity[J]. Oncogene, 2015,34: 3251- 3263.

[43] Hartman ML,Czyz M. MITF in melanoma: mechanisms behind its expression and activity[J]. Cell Mol Life Sci, 2015,72: 1249- 1260.

[44] Mezzanotte JJ, Hill V, Schmidt ML, et al. RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells[J]. Epigenetics,2014,9: 1496- 1503.

[45] Helmbold P, Richter AM, Walesch S, et al. RASSF10 promoter hypermethylation is frequent in malignant melanoma of the skin but uncommon in nevus cell nevi[J]. J Invest Dermatol, 2012, 132: 687- 694.

[46] Chen H, Zheng Z, Kim KY, et al. Hypermethylation and downregulation of glutathione peroxidase 3 are related to pathogenesis of melanoma[J]. Oncol Rep,2016, 36: 2737- 2744.

[47] Falzone L, Salemi R, Travali S, et al. MMP- 9 overexpression is associated with intragenic hypermethylation of MMP9 gene in melanoma[J]. Aging (Albany NY), 2016, 8: 933- 944.

[48] Gao L, van den Hurk K, Nsengimana J, et al. Prognostic significance of promoter hypermethylation and diminished gene expression of SYNPO2 in melanoma[J]. J Invest Dermatol,2015,135: 2328- 2331.

[49] Muthusamy V, Duraisamy S, Bradbury CM, et al. Epigenetic silencing of novel tumor suppressors in malignant melanoma[J]. Cancer Res, 2006, 66: 11187- 11193.

[50] Curry JL, Richards HW, Huttenbach YT, et al. Different expression patterns of p27 and p57 proteins in benign and malignant melanocytic neoplasms and in cultured human melanocytes[J]. J Cutan Pathol,2009,36: 197- 205.

[51] Liu W, Luo Y, Dunn JH, et al. Dual role of apoptosis-associated speck-like protein containing a CARD (ASC) in tumorigenesis of human melanoma[J]. J Invest Dermatol,2013, 133: 518- 527.

[52] Koga Y, Pelizzola M, Cheng E, et al. Genome-wide screen of promoter methylation identifies novel markers in melanoma[J]. Genome Res,2009,19: 1462- 1470.

[53] Venza M, Visalli M, Biondo C, et al. Epigenetic marks responsible for cadmium-induced melanoma cell overgrowth[J]. Toxicol In Vitro, 2015, 29: 242- 250.

[54] Furuta J, Umebayashi Y, Miyamoto K, et al. Promoter methy-lation profiling of 30 genes in human malignant melanoma[J]. Cancer Sci,2004, 95: 962- 968.

[55] Liu S, Ren S, Howell P, et al. Identification of novel epigene-tically modified genes in human melanoma via promoter methy-lation gene profiling[J]. Pigment Cell Melanoma Res,2008, 21: 545- 558.

[56] Das AM, Seynhaeve AL, Rens JA, et al. Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration[J]. Angiogenesis,2014,17: 163- 177.

[57] Schinke C, Mo Y, Yu Y, et al. Aberrant DNA methylation in malignant melanoma[J]. Melanoma Res, 2010, 20: 253- 265.

[58] McGuinness C, Wesley UV. Dipeptidyl peptidase IV (DPPIV),a candidate tumor suppressor gene in melanomas is silenced by promoter methylation[J]. Front Biosci,2008, 13: 2435- 2443.

[59] Matic IZ, Ethordic M, Grozdanic N, et al. Serum activity of DPPIV and its expression on lymphocytes in patients with melanoma and in people with vitiligo[J]. BMC Immunol,2012, 13: 48.

[60] Conway K, Edmiston SN, Khondker ZS, et al. DNA-methylation profiling distinguishes malignant melanomas from benign nevi[J]. Pigment Cell Melanoma Res, 2011,24: 352- 360.

[61] Ekstrom EJ, Sherwood V, Andersson T. Methylation and loss of Secreted Frizzled-Related Protein 3 enhances melanoma cell migration and invasion[J]. PLoS One,2011, 6: e18674.

[62] Tokita T, Maesawa C, Kimura T, et al. Methylation status of the SOCS3 gene in human malignant melanomas[J]. Int J Oncol, 2007, 30: 689- 694.

[63] Fang S, Liu B, Sun Q, et al. Platelet factor 4 inhibits IL- 17/Stat3 pathway via upregulation of SOCS3 expression in melanoma[J]. Inflammation,2014, 37: 1744- 1750.

[64] Bonazzi VF, Nancarrow DJ, Stark MS, et al. Cross-platform array screening identifies COL1A2, THBS1, TNFRSF10D and UCHL1 as genes frequently silenced by methylation in melanoma[J]. PLoS One,2011,6: e26121.

[65] Furuta J, Kaneda A, Umebayashi Y, et al. Silencing of the thrombomodulin gene in human malignant melanoma[J]. Melanoma Res,2005, 15: 15- 20.

[66] Mori T, O’Day SJ, Umetani N, et al. Predictive utility of circulating methylated DNA in serum of melanoma patients receiving biochemotherapy[J]. J Clin Oncol,2005, 23: 9351- 9358.

[67] Lian CG, Xu Y, Ceol C, et al. Loss of 5-hydroxymethylcy-tosine is an epigenetic hallmark of melanoma[J]. Cell,2012,150: 1135- 1146.

[68] Gambichler T, Sand M, Skrygan M. Loss of 5-hydroxymethylcytosine and ten-eleven translocation 2 protein expression in malignant melanoma[J]. Melanoma Res,2013,23: 218- 220.

[69] Lee JJ, Cook M, Mihm MC, et al. Loss of the epigenetic mark, 5-Hydroxymethylcytosine, correlates with small cell/nevoid subpopulations and assists in microstaging of human melanoma[J]. Oncotarget,2015, 6: 37995- 38004.

[70] Lee JJ, Granter SR, Laga AC, et al. 5-Hydroxymethyl-cytosine expression in metastatic melanoma versus nodal nevus in sentinel lymph node biopsies[J]. Mod Pathol,2015, 28: 218- 229.

[71] Pavlova O, Fraitag S, Hohl D. 5-hydroxymethylcytosine expression in proliferative nodules arising within congenital nevi allows differentiation from malignant melanoma[J]. J Invest Dermatol, 2016, 136: 2453- 2461.

[72] Saldanha G, Joshi K, Lawes K, et al. 5-Hydroxymethylcyto-sine is an independent predictor of survival in malignant melanoma[J]. Mod Pathol,2017,30: 60- 68.

[73] Thienpont B, Galle E, Lambrechts D. TET enzymes as oxygen-dependent tumor suppressors: exciting new avenues for cancer management[J]. Epigenomics,2016,8: 1445- 1448.

[74] Gong F, Guo Y, Niu Y, et al. Epigenetic silencing of TET2 and TET3 induces an EMT-like process in melanoma[J]. Oncotarget, 2017,8: 315- 328.

[75] Sigalotti L, Covre A, Fratta E, et al. Epigenetics of human cutaneous melanoma: setting the stage for new therapeutic strategies[J]. J Transl Med,2010, 8: 56.

[76] Ye Y, Jin L, Wilmott JS, et al. PI (4,5)P2 5-phosph-atase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma[J]. Nat Commun, 2013, 4: 1508.

[77] Jazirehi AR,Arle D. Epigenetic regulation of the TRAIL/Apo2L apoptotic pathway by histone deacetylase inhibitors: an attractive approach to bypass melanoma immunotherapy resistance[J]. Am J Clin Exp Immunol, 2013,2: 55- 74.

[78] Zhang XD, Gillespie SK, Borrow JM, et al. The histone deacetylase inhibitor suberic bishydroxamate regulates the expression of multiple apoptotic mediators and induces mitochondria-dependent apoptosis of melanoma cells[J]. Mol Cancer Ther,2004,3: 425- 435.

[79] Bachmann IM, Halvorsen OJ, Collett K, et al. EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast[J]. J Clin Oncol,2006,24: 268- 273.

[80] Fan T, Jiang S, Chung N, et al. EZH2-dependent suppres-sion of a cellular senescence phenotype in melanoma cells by inhibition of p21/CDKN1A expression[J]. Mol Cancer Res,2011,9: 418- 429.

[81] Mahmoud F, Shields B, Makhoul I, et al. Role of EZH2 histone methyltrasferase in melanoma progression and metastasis[J]. Cancer Biol Ther, 2016,17: 579- 591.

[82] Sengupta D, Byrum SD, Avaritt NL, et al. Quantitative histone mass spectrometry identifies elevated histone H3 lysine 27 (Lys27) trimethylation in melanoma[J]. Mol Cell Proteomics, 2016, 15: 765- 775.

[83] Ceol CJ, Houvras Y, Jane-Valbuena J, et al. The histone methyltransferase SETDB1 is recurrently amplified in melano-ma and accelerates its onset[J]. Nature, 2011, 471: 513- 517.

[84] Miura S, Maesawa C, Shibazaki M, et al. Immunohistochemistry for histone h3 lysine 9 methyltransferase and demethylase proteins in human melanomas[J]. Am J Dermatopathol, 2014, 36: 211- 216.

[85] Kostaki M, Manona AD, Stavraka I, et al. High-frequency p16(INK) (4A) promoter methylation is associated with histone methyltransferase SETDB1 expression in sporadic cutaneous melanoma[J]. Exp Dermatol, 2014, 23: 332- 338.

[86] Jiang L, Lv X, Li J, et al. The status of microRNA- 21 expression and its clinical significance in human cutaneous malignant melanoma[J]. Acta Histochem, 2012,114: 582- 588.

[87] Zhang J, Lu L, Xiong Y, et al. MLK3 promotes melanoma proliferation and invasion and is a target of microRNA- 125b[J]. Clin Exp Dermatol,2014, 39: 376- 384.

[88] Vergani E, Di Guardo L, Dugo M, et al. Overcoming melanoma resistance to vemurafenib by targeting CCL2-induced miR- 34a, miR- 100 and miR- 125b[J]. Oncotarget,2016, 7: 4428- 4441.

[89] Levati L, Pagani E, Romani S, et al. MicroRNA- 155 targets the SKI gene in human melanoma cell lines[J]. Pigment Cell Melanoma Res,2011,24: 538- 550.

[90] Liu S, Tetzlaff MT, Liu A, et al. Loss of microRNA- 205 expression is associated with melanoma progression[J]. Lab Invest, 2012,92: 1084- 1096.

[91] Levy C, Khaled M, Iliopoulos D, et al. Intronic miR- 211 assumes the tumor suppressive function of its host gene in melanoma[J]. Mol Cell, 2010, 40: 841- 849.

[92] Rinn JL, Chang HY. Genome regulation by long noncoding RNAs[J]. Annu Rev Biochem,2012, 81: 145- 166.

[93] Wapinski O, Chang HY. Long noncoding RNAs and human disease[J]. Trends Cell Biol, 2011,21: 354- 361.

[94] Li Z, Chao TC, Chang KY, et al. The long noncoding RNA THRIL regulates TNFalpha expression through its interaction with hnRNPL[J]. Proc Natl Acad Sci USA,2014, 111: 1002- 1007.

[95] Guo L, Yao L, and Jiang Y. A novel integrative approach to identify lncRNAs associated with the survival of melanoma patients[J]. Gene,2016,585: 216- 220.

[96] Flockhart RJ, Webster DE, Qu K, et al. BRAFV600E remodels the melanocyte transcriptome and induces BANCR to regulate melanoma cell migration[J]. Genome Res,2012, 22: 1006- 1014.

[97] Li R, Zhang L, Jia L, et al. Long non-coding RNA BANCR promotes proliferation in malignant melanoma by regulating MAPK pathway activation[J]. PLoS One, 2014,9: e100893.

[98] Khaitan D, Dinger ME, Mazar J, et al. The melanoma-upre-gulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion[J]. Cancer Res, 2011, 71: 3852- 3862.

[99] Gupta RA, Shah N, Wang KC, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis[J]. Nature,2010, 464: 1071- 1076.

[100] Lessard L, Liu M, Marzese DM, et al. The CASC15 long intergenic noncoding RNA locus is involved in melanoma progression and phenotype switching[J]. J Invest Dermatol,2015, 135: 2464- 2474.

[101] Tian Y, Zhang X, Hao Y, et al. Potential roles of abnormally expressed long noncoding RNA UCA1 and Malat- 1 in metastasis of melanoma[J]. Melanoma Res,2014,24: 335- 341.