劉曦宇 裴長艷 崔有斌

(吉林大學白求恩第一醫(yī)院胸部外科，吉林 長春 130021)

表觀遺傳學是目前生物學研究領(lǐng)域最具發(fā)展前景的學科〔1～4〕?；蜃陨淼母淖兒捅碛^遺傳基因的改變都會通過復雜的信號轉(zhuǎn)導,單獨或共同的引起有害基因突變率的增加。表觀遺傳學通過幾個機制控制基因的表達:DNA甲基化；組蛋白修飾；非編碼RNA的表觀遺傳調(diào)控〔5,6〕。目前研究最廣泛的是DNA甲基化〔7〕。實驗表明DNA重復序列的甲基化，可能會導致細胞染色體缺失或重組，從而造成遺傳不穩(wěn)定。因此人類基因甲基化增加染色質(zhì)的不穩(wěn)定性，導致腫瘤發(fā)展；同時特殊位點CpG島啟動子的甲基化，可以沉默腫瘤抑癌基因，有一定的致癌作用〔8〕。表觀遺傳學與飲食、藥物等外界環(huán)境有一定關(guān)系，當不利的環(huán)境因素作用于人體時，表觀改變使染色質(zhì)結(jié)構(gòu)重塑，繼而促進腫瘤等疾病的發(fā)生。

食管鱗狀細胞癌(ESCC)是遺傳、環(huán)境及飲食等多種因素共同作用的多基因、多途徑、多階段改變的疾病過程，是我國最常見的食管癌類型，發(fā)病人群以中老年為主，是最具侵犯性的腫瘤之一〔9〕，與中晚期ESCC不同，早期ESCC 5年生存率>90%〔10〕。盡管目前的診療技術(shù)不斷發(fā)展，但由于多種原因存在早期診斷困難，進而不能及時得到治療和完整切除腫瘤。因此尋找敏感度和特異度高的無創(chuàng)性診斷技術(shù)或指標是提高ESCC生存率的重要途徑。在治療效果有限的情況下促使尋找一種新的策略去治療ESCC，尤其是針對一些特定的因子去進行早期診斷。大量研究證明，在表觀遺傳變化上，DNA的甲基化在ESCC過程中起到至關(guān)重要的作用。由于表觀遺傳變化是一個可逆的過程，因此，在ESCC的早期診斷和治療上將會有更廣泛的應用。

1 人類腫瘤的DNA甲基化改變

甲基化是主要的表觀遺傳修飾中的一種，其通過腫瘤抑制基因的啟動子區(qū)抑制轉(zhuǎn)錄，甲基化通常發(fā)生在啟動子區(qū)的CpG島，且與基因失活有關(guān)。整體的甲基化也通過不同機制與癌癥的發(fā)生和發(fā)展有關(guān)〔11〕。已知DNA甲基化通過至少3個獨立的DNA甲基轉(zhuǎn)移酶(DNMT1)、DNMT3a、DNMT3b之間復雜的相互作用而對環(huán)境因子做出反應，其共同作用在于生成和維護遺傳基因甲基化模式。DNMT1在DNA復制后將甲基化模式轉(zhuǎn)移給一條新的合成鏈，因此經(jīng)常作為“維持”甲基轉(zhuǎn)移酶被提及〔12〕, DNMT3a 和DNMT3b顯示非甲基化和半甲基化DNA特性，催化新的甲基化位點形成〔13〕。

DNA的甲基化發(fā)生在多數(shù)基因序列上，包括重復序列和逆轉(zhuǎn)錄轉(zhuǎn)座子，因此導致基因的不穩(wěn)定性〔14〕。DNA重復序列的甲基化可能引起有絲分裂重組，進而導致基因缺失或異位，并同時促進染色體重組〔15〕。另外，異常的DNA甲基化可活化一些原癌基因和印跡缺失，如Angelman綜合征，普拉德-威利綜合征，脆弱型X染色體癥。

目前表觀遺傳學公認的導致腫瘤發(fā)生的原因是CpG島的甲基化和腫瘤抑癌基因的沉默。CpG核酸不均勻的分布在整個基因序列，但是大量集中分布在重復序列，如逆轉(zhuǎn)錄轉(zhuǎn)座子、rDNA、CpG島等。約60%哺乳動物在蛋白編碼基因的啟動子區(qū)域含有CpG島，其甲基化導致腫瘤可能通過三種機制：胞嘧啶甲基化后形成5-甲基胞嘧啶，促進基因突變〔16,17〕；異常的DNA甲基化導致相關(guān)的等位基因缺失〔18〕；DNA啟動子區(qū)域甲基化導致腫瘤抑癌基因沉默或失活〔19〕。

2 ESCC中甲基化的改變

20年前，腫瘤組織內(nèi)的基因序列甲基化第1次被發(fā)現(xiàn)〔20〕，后期發(fā)現(xiàn)甲基化可能與致癌作用相關(guān)〔21〕。然而，甲基化在ESCC致病過程中的機制仍知之甚少，目前比較公認的有以下幾種機制：

2.1DNA序列的甲基化 基因啟動子的甲基化和相應位點的缺失是目前世界公認的腫瘤標志，DNA甲基化已經(jīng)成為ESCC最有前途的生物學治療靶點及檢測標志物，目前有大量的研究證明了多種基因的表觀遺傳學改變與ESCC的發(fā)生發(fā)展過程密切相關(guān)〔22～63〕。

人類基因序列大約17%由逆轉(zhuǎn)錄轉(zhuǎn)座子構(gòu)成，其甲基化水平是重要的DNA甲基化標志，在ESCC中可達25%～92%，逆轉(zhuǎn)錄轉(zhuǎn)座子的甲基化高表達同樣預示預后不良〔64〕。需要進一步的研究證實各因素之間的關(guān)系，全基因組芯片測序技術(shù)將會更廣泛的應用到抗腫瘤治療中。

2.2腫瘤抑制基因的甲基化 食管癌患者腫瘤組織和血漿中發(fā)現(xiàn)一些抑癌基因啟動子區(qū)異常甲基化，目前已經(jīng)被確認為多種腫瘤早期的特征性基因改變，其中甲基化率較高的抑癌基因有如下幾種：腺瘤性結(jié)腸息肉病基因(APC)通過與微管結(jié)合，調(diào)節(jié)β-蛋白水平，間接調(diào)節(jié)細胞增殖，在ESCC中，研究顯示有55%～80%的APC位點缺失〔65〕和少量的基因突變〔66〕，APC啟動子區(qū)域的甲基化達到27%～46%〔22〕，并與淋巴結(jié)轉(zhuǎn)移有相關(guān)性〔23〕。

細胞周期調(diào)控基因P16，在細胞增殖的調(diào)控中，p16 蛋白與細胞周期蛋白(cyclin D1)競爭與Cyclin D1依賴性激酶家族(CDK4/CDK6)的結(jié)合，發(fā)揮負性調(diào)節(jié)作用，在ESCC中甲基化頻率達到57.8%～81.6%〔67,68〕，并與腫瘤分化、分期和淋巴結(jié)轉(zhuǎn)移有關(guān)。上皮細胞鈣黏蛋白基因(CDH1),在ESCC中甲基化表達率達14%～61%，CDH1的缺失與腫瘤的侵襲、轉(zhuǎn)移和預后不良明顯相關(guān)〔69,70〕。抑癌基因(CNKN2A)，在ESCC甲基化程度達到19%～88%〔26，71，72〕，其失活與腫瘤的侵襲、轉(zhuǎn)移密切相關(guān)。脆性組氨酸三聯(lián)體(FHIT)，在ESCC甲基化程度達到14%～85%〔50，73，74〕，其表達缺失與吸煙有關(guān)〔37〕，F(xiàn)HIT的缺失與早期淋巴轉(zhuǎn)移和不良預后明顯相關(guān)。視黃酸受體-β(RARβ)參與腫瘤生長和誘導凋亡，在正常食管上皮細胞中有12%陽性表達，ESCC組織中約70%表達〔75〕早期ESCC中也達到67%甲基化〔50〕。Ras相關(guān)區(qū)域家族1(RASSF1) ，正常組織中RASSF1啟動子甲基化只有3%～4%，在ESCC組織中達13%～53%，與腫瘤分化程度相關(guān)〔76～78〕。Runt 相關(guān)轉(zhuǎn)錄因子3(RUNX3)，參與TGF-β上皮細胞生長的負調(diào)控，其正常編碼產(chǎn)物RUNX蛋白與絲/蘇氨酸激酶受體(Smad)形成復合物，共同調(diào)節(jié)相關(guān)靶基因的轉(zhuǎn)錄，RUNX3表達缺失將導致Smad蛋白功能的限制TGF-β的生長抑制作用，進而促使腫瘤細胞擺脫〔52〕，ESCC 組織RUNX3 啟動子甲基化頻率為64.3%并與淋巴結(jié)轉(zhuǎn)移有相關(guān)性〔53〕。

2.3ESCC的外周血DNA甲基化 多年前已有研究表明腫瘤細胞在增殖的過程中可以釋放大量游離的 DNA 到外周血循環(huán)，并富集于血清或血漿中,腫瘤患者血清中DNA可能來源于腫瘤DNA，其甲基化程度明顯高于正常人〔79，80〕,但甲基化比例卻低于ESCC腫瘤組織，近年研究通過分析ESCC 患者外周血游離DNA以及配對組織DNA 中多種抑癌基因啟動子的甲基化狀態(tài)，發(fā)現(xiàn)外周血游離DNA 高甲基化是ESCC 特異性的早期診斷、預后評估的潛在分子標志物，為發(fā)展臨床診療新策略提供理論基礎(chǔ)。

3 基因芯片技術(shù)在甲基化檢測的應用

目前大部分關(guān)于ESCC甲基化方面的研究都是基于特定基因，缺乏高通量、大樣本、高靈敏度的檢測，芯片技術(shù)的發(fā)展將這一瓶頸打破，克服了以往檢測手段操作復雜、成本高、效率低等缺點?；蛐酒膬?yōu)點：(1)基因芯片可一次性分析數(shù)萬個靶基因片段，通過對比可獲得數(shù)十個有表達差異的基因;(2)通過堿基互補原理，特異性結(jié)合，準確率高；(3)通過計算機讀取，自動化程度高，數(shù)據(jù)分析效率明顯提高?；驕y序后，通過比較分析不同個體之間的序列差異，可以發(fā)現(xiàn)功能基因，并可以解碼每個核酸的生物學意義，識別與疾病相關(guān)的基因，從而達到預防、診斷和治療的目的。

4 DNA甲基化可成為治療新靶點

與其他不可逆轉(zhuǎn)的基因改變不同，DNA的甲基化是一個可逆的過程，這一機制可以作為分子治療腫瘤的潛在位點。由DNMT1、DNMT3a、DNMT3b 3種酶催化，假設(shè)DNMT酶蛋白表達被抑制，會造成沉默基因的激活，抑癌基因的增殖，促進細胞凋亡，增加細胞對化療藥物的敏感度，因此DNMT抑制劑是目前的研究熱點。但是到目前為止還沒有專門針對ESCC治療有效的藥物報導。一些天然成份(茶多酚)抑制DNMT1的活性部位，導致甲基化沉默基因的激活，從而達到抗癌目的〔81〕。這些自然元素可以和常規(guī)治療相結(jié)合，為ESCC的治療打開一個新的領(lǐng)域。

5 結(jié)論與展望

表觀遺傳學是生物學和腫瘤研究方面迅速擴張的領(lǐng)域，其在ESCC預防、診斷、治療方面具有巨大的潛力及深遠影響，同時基因組測序技術(shù)的發(fā)展推動了生物學各個領(lǐng)域的發(fā)展，尤其測序成本迅速降低，實現(xiàn)高通量的多基因位點DNA甲基化聯(lián)合檢測成為可能。ESCC的早期發(fā)現(xiàn)對癌癥患者的有效治療非常重要，許多ESCC患者臨床癥狀出現(xiàn)較晚，并且活體取樣檢測也存在一定的局限性，嚴重影響了癌癥的早期診斷和對病人有效的治療。雖然腫瘤的發(fā)生發(fā)展是一個復雜的過程，但通過芯片技術(shù)篩選出腫瘤早期發(fā)生甲基化改變的特異性位點是可能實現(xiàn)的，將外周血DNA甲基化測定和基因芯片檢測相結(jié)合，給早期ESCC診斷提供了新的手段，同時對抑制甲基化方法的不斷探索深入，對ESCC患者的診斷和治療效果將會有巨大的積極意義。

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