膠質(zhì)瘤中的表觀遺傳學(xué)：一場基因革命

祁 婧，閔 彬，涂艷陽

(1空軍軍醫(yī)大學(xué)唐都醫(yī)院實驗外科,陜西 西安710038；2空軍工程大學(xué)門診部,陜西西安710051)

0 引言

神經(jīng)膠質(zhì)瘤是滲透性的腦瘤,最常見的是星形細胞瘤和少突神經(jīng)膠質(zhì)瘤,被分為低級別(WHO等級Ⅰ和Ⅱ)和高級別腫瘤(WHO等級Ⅲ和Ⅳ)。膠質(zhì)母細胞瘤(Ⅳ級星形細胞腫瘤)是最致命且最具有破壞性的神經(jīng)膠質(zhì)瘤。盡管經(jīng)過幾十年的研究,膠質(zhì)母細胞瘤和高級別神經(jīng)膠質(zhì)瘤的預(yù)后仍然很差。這強調(diào)了闡明腫瘤發(fā)病機制的重要性。近年來在膠質(zhì)瘤分子遺傳學(xué)方面取得了許多新進展,并已應(yīng)用于膠質(zhì)瘤的分型中,其中許多基因和分子改變可導(dǎo)致細胞代謝的顯著變化。

傳統(tǒng)分子靶向治療方法主要集中在諸如點突變、基因缺失和重排等基因的結(jié)構(gòu)變化,這些改變參與了膠質(zhì)瘤的發(fā)生及演進,對其診斷、治療及預(yù)后判斷也具有重要的作用。例如,膠質(zhì)瘤中出現(xiàn)的各種基因改變(如EGFR擴增、PTEN損失、PDGFRA擴增)可導(dǎo)致受體酪氨酸激酶信號增強和PI3K/AKT通路的失調(diào),從而刺激葡萄糖攝取和有氧糖酵解。在Ⅱ級和Ⅲ級星形細胞瘤、少突神經(jīng)膠質(zhì)瘤和膠質(zhì)母細胞瘤中70%都存在NADP+依賴性酶異檸檬酸脫氫酶1(socitrate dehydrogenase 1,IDH1)的突變。IDH1催化氧化胞質(zhì)內(nèi)異檸檬酸脫羧生成 α-酮戊二酸(α-KG)。IDH1突變改變了細胞代謝,其代謝產(chǎn)物2-羥戊二酸(2-HG)的積累會在腫瘤的發(fā)生發(fā)展中發(fā)揮作用.

轉(zhuǎn)錄/翻譯水平上的基因表達的調(diào)控機制是癌癥研究中最新興的領(lǐng)域。最近的研究［8－9］表明惡性轉(zhuǎn)化是由遺傳變異和表觀遺傳學(xué)改變的復(fù)雜相互作用所致的,從而影響各種細胞生物學(xué)過程的變化,包括細胞增殖和侵襲、DNA修復(fù)、凋亡、血管生成和細胞周期調(diào)控,最終導(dǎo)致腫瘤形成。

表觀遺傳學(xué)的現(xiàn)象很多,已知的有DNA甲基化、基因組印記、母體效應(yīng)、基因沉默、核仁顯性、休眠轉(zhuǎn)座子激活以及RNA編輯等。DNA甲基化是惡性膠質(zhì)瘤中研究最廣泛的表觀遺傳現(xiàn)象。組蛋白是核小體形成過程中所必需的蛋白質(zhì)。每一個核小體由約147個DNA堿基對纏繞在組蛋白八聚體上,組蛋白八聚體由H2A、H2B、H3和H4組成。組蛋白氨基酸的尾巴可以接受各種翻譯后修飾如乙?；?、甲基化、磷酸化、泛素化和精氨酸(R)和賴氨酸(K)殘基類泛素化。組蛋白賴氨酸殘基的乙?；饔猛ǔ＜せ钷D(zhuǎn)錄,而甲基化(H3K9、H3K27、H4K20、H3K4 甲基化)可以激活或抑制轉(zhuǎn)錄相關(guān)因子。

1 DNA甲基化

研究最多的表觀遺傳修飾是胞嘧啶甲基化,在哺乳動物細胞中,DNA甲基化主要發(fā)生在胞嘧啶殘基上,其次是鳥嘌呤。DNA甲基化是由DNMT(DNA甲基轉(zhuǎn)移酶)家族的催化轉(zhuǎn)移S-腺苷甲硫氨酸的甲基到DNA上。到目前為止,五個DNMT家族成員已被確定,分別為 DNMT1、DNMT2、DNMT3a、DNMT3b 和DNMT3L。DNA甲基化與轉(zhuǎn)錄活性相關(guān)。眾所周知,在人類的癌癥中DNA甲基化譜在腫瘤發(fā)生和進展中扮演著重要角色［15－16］。

人神經(jīng)膠質(zhì)瘤表現(xiàn)出甲基化模式的腫瘤典型變化［17－20］。低甲基化主要發(fā)生在 DNA 重復(fù)區(qū)域,并可通過激活致癌基因和增加基因組不穩(wěn)定性來促進腫瘤生長。高甲基化主要發(fā)生在基因啟動子CpG島上,參與腫瘤形成和進展的過程,這些基因大都與腫瘤抑制［21－22］、 DNA 修復(fù)［23］、 細胞周期調(diào)控［24］、 凋亡［25－26］、侵襲［27－28］和遷移［29］相關(guān)(表 1)。 有趣的是,膠質(zhì)瘤分級不同甲基化模式也不同,在膠質(zhì)瘤WHO的Ⅱ級,Ⅲ級和Ⅳ級之間基因的甲基化的狀態(tài)也呈現(xiàn)出明顯差異［30］。

在癌癥基因組圖譜(the cancer genome atlas,TCGA)項目的框架內(nèi),Noushmehr等［6］研究甲基化譜分析確定了GBM腫瘤表型,其特征在于大量基因位點的協(xié)同高甲基化,被稱為G-CIMP。G-CIMP與延長生存期以及基因表達譜(突變表達模式)有關(guān)［31］。此外,發(fā)現(xiàn)IDH突變導(dǎo)致酶活性的變化,α-KG產(chǎn)生減少,產(chǎn)生代謝物2-羥基戊二酸(2-HG),其競爭性地抑制調(diào)節(jié)DNA和組蛋白甲基化的酶的活性(α-KG依賴性雙加氧酶),包括組蛋白脫甲基酶［32］和TET5mC羥化酶家族［33－35］。 TET 蛋白能通過轉(zhuǎn)化 5-甲基胞嘧啶(5mC)至5-羥甲基胞嘧啶(5hmC)改變DNA甲基化狀態(tài)。5hmC的生物學(xué)功能尚未得到確鑿的闡明。與正常腦相比,在人類膠質(zhì)瘤中,5hmC顯著減少,并且已經(jīng)有研究顯示5hmC水平與細胞增殖之間呈現(xiàn)反比關(guān)系［36－37］。這些發(fā)現(xiàn)揭示了基因調(diào)節(jié)的另一種水平,并證明與膠質(zhì)瘤發(fā)生中遺傳和表觀遺傳密切相關(guān)［38－39］。

表1 人類膠質(zhì)瘤中主要的表觀遺傳改變

在過去十年中,關(guān)于DNA甲基化過程的研究發(fā)現(xiàn)了許多腫瘤重要的生物標(biāo)志物。O6-甲基鳥嘌呤-DNA甲基轉(zhuǎn)移酶(methylguanine methyl transferase,MGMT)是一種DNA修復(fù)酶,其去除鳥嘌呤O6位置的烷基加合物,從而保護正常細胞免受致癌物質(zhì)的侵害,相反的是,MGMT也可以來保護接受化療的腫瘤細胞。MGMT表達可以通過啟動子甲基化進行表觀遺傳沉默,這種情況出現(xiàn)在35%～45%的惡性膠質(zhì)瘤和80%的WHOⅡ級膠質(zhì)瘤中［40－41］。啟動子甲基化狀態(tài)已被確定為惡性膠質(zhì)瘤患者進行烷基化劑化療中治療效果明顯且獨立的預(yù)測因子。通常與未甲基化的MGMT啟動子相比,存在MGMT啟動子甲基化形式的患者用替莫唑胺治療效果更加顯著［42－43］。這說明MGMT啟動子甲基化狀態(tài)已被確定為神經(jīng)腫瘤學(xué)的重要臨床標(biāo)志物。然而不是所有MGMT啟動子甲基化的患者在替莫唑胺治療后均有顯著的治療效果。在這些患者中,已經(jīng)發(fā)現(xiàn)MGMT啟動子甲基化和MGMT的mRNA表達不一致,不論MGMT啟動子甲基化或是未甲基化,在25%的膠質(zhì)母細胞瘤中檢測到MGMT的mRNA表達異常；攜帶低轉(zhuǎn)錄活性的MGMT患者具有更好的治療效果,這一結(jié)果與MGMT啟動子甲基化結(jié)果正好相反,這種不一致的基本機制尚不清楚。我們假設(shè),MGMT低表達水平與未甲基化的啟動子組合的情況可能由轉(zhuǎn)錄物不穩(wěn)定和/或轉(zhuǎn)錄抑制因子如miRNA調(diào)節(jié)或組蛋白修飾引起［44］。

2 組蛋白修飾

染色質(zhì)是細胞核中DNA和組蛋白的縮合形式。在真核生物中,染色質(zhì)由147個堿基對的DNA組成,緊緊纏繞在兩個拷貝的四個核心組蛋白H2A,H2B,H3和H4八聚體周圍的。

所得到的核小體是染色質(zhì)的基礎(chǔ)重復(fù)單元［45］。由于每個核心組蛋白具有從核小體突出的氨基末端“尾”,組蛋白,特別是其尾巴可能存在一定數(shù)量的翻譯后修飾。組蛋白修飾包括乙?；⒓谆土姿峄?但也存在研究較少的修飾,如泛素化、ADP核糖基化、脫氨基和脯氨酸異構(gòu)化［46］。這些組蛋白修飾中的每一種都能夠影響染色質(zhì)結(jié)構(gòu),從而導(dǎo)致DNA修復(fù)以及基因轉(zhuǎn)錄的改變。組蛋白修飾可以廣泛地分為主動標(biāo)記和被動標(biāo)記。特別是組蛋白乙?；图谆谥掳C制中發(fā)揮顯著作用［15,47］。

賴氨酸殘基的乙?；山M蛋白乙酰轉(zhuǎn)移酶(HATs)和組蛋白脫乙酰酶(HDAC)的相反作用調(diào)節(jié)。乙酰化中和賴氨酸殘基的正電荷,從而削弱DNA和組蛋白尾部之間的鍵。因此,組蛋白乙?；c轉(zhuǎn)錄激活相關(guān),而脫乙酰化通常與抑制轉(zhuǎn)錄有關(guān)。組蛋白甲基化主要發(fā)生在賴氨酸和精氨酸的側(cè)鏈上,其影響轉(zhuǎn)錄機制的效應(yīng)蛋白的活性。組蛋白甲基化可以激活(例如 H3K4me2、H3K4me3)或抑制(H3K9me2,H3K27me3)轉(zhuǎn)錄,這取決于各自的甲基化位點［48－50］。

組蛋白表達水平的改變也可能在膠質(zhì)瘤發(fā)生中發(fā)揮作用。這些改變包括參與組蛋白修飾的基因的異常表達以及各基因的組蛋白修飾模式的變化(表1)。組蛋白水平的畸變來源于調(diào)節(jié)基因突變,如GBM(包括HDAC2和HDAC9),組蛋白去甲基化酶(JMJD1A和 JMJD1B),組蛋白甲基轉(zhuǎn)移酶(SET7、SETD7、MLL、MLL3 和 MLL4)［5］。 此外,HDAC 的表達水平的改變已被報道與腫瘤復(fù)發(fā)和進展相關(guān)(HDAC1、HDAC2 和 HDAC3)［51－52］。 在幾項研究中已經(jīng)報道了組蛋白修飾調(diào)節(jié)單個基因。例如,抑制腫瘤抑制因子RRP22和細胞周期調(diào)節(jié)因子p21的表達以及促增殖轉(zhuǎn)錄因子HOXA9的增強表達與組蛋白修飾模式的改變相關(guān)［53－55］。 但是,組蛋白修飾實際功能在膠質(zhì)瘤中的作用及其作為生物標(biāo)志物和/或治療靶標(biāo)的潛力仍有待充分闡明。

3 MicroRNAs

近來已發(fā)現(xiàn)非編碼RNA在基因表達的表觀遺傳調(diào)控中起重要作用［56－57］。其中miRNA是約22個核苷酸(nt)長度的雙鏈RNA分子,起源于人類基因組轉(zhuǎn)錄物前體。通過結(jié)合目標(biāo)mRNA的3’-UTR內(nèi)的特異性識別序列,抑制翻譯或mRNA降解調(diào)節(jié)基因表達［58－59］。 目標(biāo)識別主要通過 miRNA 的 5’區(qū)域的8個核苷酸短序列的堿基配對進行介導(dǎo)［60］。雖然一些miRNA調(diào)節(jié)特定的目標(biāo),但是來自多個研究的證據(jù)表明某些關(guān)鍵的miRNA可以調(diào)節(jié)高達幾百個靶基因,并且許多類型的 miRNA 協(xié)同調(diào)節(jié)其靶標(biāo)［61－62］。研究人員使用計算預(yù)測方法和不同測序技術(shù)相結(jié)合等技術(shù)手段,已經(jīng)確定了大量的調(diào)節(jié)分子［63－64］。 目前發(fā)布的mirbase數(shù)據(jù)庫,存在超過140種物種中含有的超過17,000個成熟miRNA序列［65］。最近的研究［66］表明大部分的轉(zhuǎn)錄組受到miRNAs調(diào)控。這些結(jié)論說明miRNA表達的調(diào)節(jié)異常與病理學(xué)特征及其預(yù)后相關(guān)聯(lián)。

已經(jīng)在許多類型的人類腫瘤中檢測出miRNA的異常表達,包括神經(jīng)膠質(zhì)瘤［67－68］。 然而,miRNA 不僅僅作為腫瘤抑制因子起作用,而且還依賴靶向mRNA的功能作為一種癌基因［69－71］。 因此,改變的 miRNA表達水平會對致癌過程產(chǎn)生重大影響。與正常細胞相比,miRNA在惡性腫瘤中的差異表達的原因尚未完全闡明。然而,miRNA的轉(zhuǎn)錄調(diào)控序列中的表觀遺傳修飾以及基因突變,基因組缺失或基因擴增等遺傳改變可能影響miRNA成熟和/或與mRNA靶標(biāo)相互作用［72－74］。

在GBM中,高通量分析已經(jīng)確定了miRNA的差異表達［75－77］。因此,miRNA被認(rèn)為是GBM多重生物學(xué)特征的重要介質(zhì),包括細胞增殖、G1/S細胞周期進程、細胞存活、細胞遷移和細胞侵襲［78］。盡管尚未闡明神經(jīng)膠質(zhì)瘤復(fù)雜網(wǎng)絡(luò)中miRNA的確切功能,但越來越多的研究集中于miRNA在神經(jīng)膠質(zhì)瘤發(fā)生和進展過程中的不同功能(表1)。例如,與正常腦相比,在神經(jīng)膠質(zhì)瘤中下調(diào)的miRNA已經(jīng)被發(fā)現(xiàn)通過直接靶向致癌基因 c-Met、Notch［79－80］、Bmi-1［76］、表皮生長因子受體［81］、受體酪氨酸激酶［82］和細胞周期成分［83］發(fā)揮抑癌作用。相反,在膠質(zhì)瘤中具有高表達的miRNA可能被確定為致癌基因,例如miR-21通過靶向基質(zhì)金屬蛋白酶的調(diào)節(jié)劑,miR-26a靶向PTEN和miR-10b 靶向細胞周期抑制劑［84－90］。

4 表觀遺傳網(wǎng)絡(luò)

表觀遺傳調(diào)節(jié)途徑通過相互作用形成復(fù)雜的調(diào)控網(wǎng)絡(luò):①miRNA本身的表達可以通過由組蛋白和/或DNA甲基化的共價修飾引起的染色質(zhì)結(jié)構(gòu)的變化來修飾［91－92］。②腫瘤可以相互利用 miRNA來靶向表觀遺傳。例如,發(fā)現(xiàn)miR-29b在急性骨髓性白血病中靶向DNMT3a和3b,miR-449a控制前列腺癌細胞中的HDAC1。在人神經(jīng)膠質(zhì)瘤中,miR-185最近被證實作為DNMT1的調(diào)節(jié)因子,其過表達導(dǎo)致整體DNA 低甲基化［93－95］。 此外,已經(jīng)發(fā)現(xiàn) miR-101 靶向組蛋白甲基轉(zhuǎn)移酶EZH2且在人類GBM中下調(diào),從而促進腫瘤生長［96－97］。③組氨酸修飾酶可能被CpG高甲基化沉默。例如,NSD1基因編碼參與染色質(zhì)調(diào)節(jié)的組蛋白甲基轉(zhuǎn)移酶,其沉默導(dǎo)致組蛋白殘基H4K20和H3K36的甲基化減少,同時又導(dǎo)致致癌基因MEIS1的活化［98］。我們需要在全基因組范圍進一步研究,以充分闡明神經(jīng)膠質(zhì)瘤中的表觀遺傳模式,這將為闡明膠質(zhì)瘤等高度異質(zhì)性腫瘤的表觀遺傳學(xué)網(wǎng)絡(luò)的復(fù)雜模式提供理論依據(jù)［99］。

5 結(jié)論與展望

分子生物標(biāo)志物的研究進展改變了目前在世界衛(wèi)生組織分類框架內(nèi)的診斷精確度,同時有利于揭示相同WHO級別但是不同預(yù)后和治療反應(yīng)的神經(jīng)膠質(zhì)瘤亞群之間的差異［2］。1p/19共缺失檢測與化療和/或放療后的預(yù)后結(jié)果相關(guān)［100－101］。 篩查 IDH1/2突變有助于區(qū)分來自彌漫性星形細胞瘤的WHOⅠ級毛細胞星形細胞瘤和室管膜瘤(不含IDH突變)。IDH突變與間變性星形細胞瘤和膠質(zhì)母細胞瘤的良好預(yù)后相關(guān)［102－103］。 鑒于在實際病例中Ⅱ～Ⅳ級神經(jīng)膠質(zhì)瘤難以進行完全腫瘤切除,不完全切除術(shù)通常不能獲得相應(yīng)的預(yù)后效果,微創(chuàng)分子表征策略的發(fā)展受到越來越多的關(guān)注［104］。最近,組織病理學(xué)診斷與新穎的分子立體定向活檢程序相結(jié)合已經(jīng)在DNA和RNA水平上實現(xiàn)高度可重復(fù)和有效的結(jié)果；MGMT啟動子甲基化,MGMT mRNA表達以及TP53突變狀態(tài)和1p/19q-狀態(tài)的信息可以從1 mm3的立體定向組織樣品中精確定義的位點收集［105－106］。

引入大規(guī)模的“二代”測序(next-generation sequencing,NGS)技術(shù)手段標(biāo)志著基因組研究革命的開始［107］。目前,全基因組、轉(zhuǎn)錄組和甲基化測序在很小的腫瘤組織樣本中也可以進行研究。NGS平臺提供了對基因組和表觀基因組更加全面的理論支持,并且基于NGS的數(shù)據(jù)的整合對于了解和鑒定神經(jīng)膠質(zhì)瘤的發(fā)生和發(fā)展的細胞內(nèi)途徑的致密網(wǎng)絡(luò)是至關(guān)重要的［108］。隨著NGS技術(shù)的成本下降和生物信息數(shù)據(jù)處理的改善,對于同一生物樣品的轉(zhuǎn)錄組和遺傳變異相結(jié)合的多個表觀遺傳修飾的基因組測序可能成為未來的臨床研究方法。在這種情況下,通過微創(chuàng)方法(如分子立體定向程序)收集腫瘤樣本將是特別有價值的。從這種方法獲得的信息可以為每名患者創(chuàng)建個性化的治療方案。將膠質(zhì)瘤中研究發(fā)現(xiàn)的表觀遺傳學(xué)現(xiàn)象應(yīng)用到膠質(zhì)瘤的早期診斷,高危人群的監(jiān)測,腫瘤風(fēng)險評估,判斷腫瘤復(fù)發(fā)情況,預(yù)測腫瘤療效和預(yù)后,開發(fā)特異新靶點藥物等方面具有很大的潛力,相信隨著檢測手段和實驗方法的日臻完善,膠質(zhì)瘤預(yù)防、診斷和治療等領(lǐng)域必將取得喜人的成果。

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