長(zhǎng)片段非編碼RNA在腫瘤領(lǐng)域研究進(jìn)展

1、長(zhǎng)片段非編碼RNA在腫瘤領(lǐng)域的研究進(jìn)展人類基因組研究顯示僅有約20000個(gè)蛋白編碼基因，占總的基因組序列不到2%1,2。 然而90% 的基因組序列都處在活躍轉(zhuǎn)錄中 3,4。研究者發(fā)現(xiàn)人類轉(zhuǎn)錄組不僅僅是蛋白編碼基因及其剪接變異體的簡(jiǎn)單組合，還存在廣泛的轉(zhuǎn)錄反義RNA、重疊RNA和非編碼RNA（non-coding RNA，ncRNA）的基因5-10。這些最初被認(rèn)為是基因組中的“暗物質(zhì)”，近期研究顯示在細(xì)胞發(fā)育和代謝中發(fā)揮重要的作用11-17。新近發(fā)現(xiàn)的長(zhǎng)鏈非編碼RNA（ long non-coding RNA，lncRNA)基因就是其中備受關(guān)注的一種，lncRNA的表達(dá)具有時(shí)空特異性，其異常調(diào)控

2、廣泛見于包括癌癥在內(nèi)的多種疾病。 ncRNA可以根據(jù)其長(zhǎng)短簡(jiǎn)單分為短鏈非編碼RNA（small ncRNA）（18-200nt）和lncRNA（200nt-100kb以上）。Small ncRNA包括我們熟知的miRNA，以及新近發(fā)現(xiàn)的轉(zhuǎn)錄起始RNA（transcription initiation RNAs，tiRNAs）等。lncRNA像mRNA一樣由RNA聚合酶生成，并且在平均長(zhǎng)度及多聚A尾和多聚A尾信號(hào)所占的百分比等方面都與mRNA分子有很大的相似之處，但是它們?nèi)狈﹂L(zhǎng)的進(jìn)化保守的開放閱讀框，沒(méi)有編碼蛋白質(zhì)的功能47,48。通常而言，lncRNA基因的表達(dá)水平要低于蛋白編碼基因52-55

3、, 而某些lncRNAs特定地表達(dá)在某些組織中21。然而,近期研究顯示新發(fā)現(xiàn)的lncRNAs可能是前面提到的轉(zhuǎn)錄組“暗物質(zhì)”中的重要組成部分56,57。與miRNA和蛋白編碼基因，某些轉(zhuǎn)錄活躍的lncRNA存在5末端三甲基化的組蛋白H3K4 和基因主體部分的三甲基化的組蛋白H3K368,58,59。目前發(fā)現(xiàn)的少量人類lncRNAs 參與了一系列的生物過(guò)程，包括表觀遺傳學(xué)調(diào)控，選擇性剪切，入核轉(zhuǎn)運(yùn)，作為結(jié)構(gòu)元件，作為小RNA前體，甚至作為mRNA衰變的調(diào)控子等4,60-70。更重要的是越來(lái)越多的研究表明lncRNA的異常調(diào)控廣泛參與了多種人類疾病，包括腫瘤的發(fā)生和發(fā)展。一、 lncRNA與腫瘤的

4、發(fā)生、發(fā)展研究者在lncRNA發(fā)現(xiàn)初期即提出腫瘤組織與正常組織中l(wèi)ncRNAs的表達(dá)存在差異的猜想，但一直缺乏有力的證據(jù)86。 隨著腫瘤轉(zhuǎn)錄組以及l(fā)ncRNAs功能學(xué)的進(jìn)展，目前已發(fā)現(xiàn)一系列在腫瘤中差異表達(dá)的lncRNAs。lncRNA參與調(diào)控多種生物功能，這些功能如基因組印跡和轉(zhuǎn)錄調(diào)控的異常在腫瘤發(fā)生中發(fā)揮重要的作用。在此，我們重點(diǎn)介紹研究較清楚的與腫瘤生物相關(guān)的一些lncRNAs。印跡lncRNA基因印跡是指來(lái)自父母一方的基因備份被轉(zhuǎn)錄后沉默87,88。癌癥的一個(gè)重要特點(diǎn)是由于印跡的丟失導(dǎo)致了基因表達(dá)的改變90,91。而其中最為人所知的基因?qū)嶋H上均為lncRNAs。H19基因編碼了一個(gè)僅表

5、達(dá)在母體等位基因的長(zhǎng)2.3kb的lncRNA。H19在脊椎動(dòng)物胚胎發(fā)育期高表達(dá)，而出生后在除了骨骼和軟骨組織外的大部分組織中表達(dá)明顯下降20,93,94。在人類腫瘤中，印跡的丟失和隨后的基因高表達(dá)現(xiàn)象十分常見。例如，H19位點(diǎn)印跡的丟失導(dǎo)致H19表達(dá)的上調(diào)廣泛見于食管癌、結(jié)腸癌、肝癌、膀胱癌及肝臟轉(zhuǎn)移瘤中95-97。H19在不同癌癥中顯示致癌或抑癌作用。H19在肝癌，膀胱癌和乳腺癌中均高表達(dá)，提示其致癌作用97-99。在結(jié)腸癌中，原癌基因轉(zhuǎn)錄因子c-Myc直接激活H19，提示H19直接參與c-Myc對(duì)下游基因表達(dá)的調(diào)控98。相反，抑癌基因和轉(zhuǎn)錄激活因子p53能下調(diào)H19的表達(dá)100,101。有

6、趣的是，也有研究顯示H19同樣具有腫瘤抑制作用104,105。Xist(X chromosome inactive specific transcript)RNA，長(zhǎng)約16500nt，能并同X染色體一起移動(dòng)，改變?nèi)旧w的結(jié)構(gòu)和活性，抑制相應(yīng)的基因轉(zhuǎn)錄，從而使x染色體失活106。XIST的表達(dá)水平與某些腫瘤的預(yù)后密切相關(guān)，如卵巢癌的治療反應(yīng)118, 但XIST在人類腫瘤的發(fā)生中的作用還未明確。腫瘤轉(zhuǎn)移HOTAIR是一個(gè)位于人類12q13.13染色體哺乳動(dòng)物HOXC位點(diǎn)的長(zhǎng)2.2 kb的基因，其編碼的lncRNA是最早發(fā)現(xiàn)與腫瘤轉(zhuǎn)移相關(guān)的lncRNA之一18。HOTAIR RNA在原發(fā)和轉(zhuǎn)移乳腺癌

7、中均高表達(dá)（高出正常組織2000倍以上）68。高表達(dá)的HOTAIR與腫瘤侵襲、轉(zhuǎn)移和患者預(yù)后不良密切相關(guān)68。如將表達(dá)HOTAIR的細(xì)胞種植到小鼠乳腺脂肪墊中,會(huì)一定程度加快原發(fā)腫瘤的生長(zhǎng)68.有趣的是，從HOX位點(diǎn)中能轉(zhuǎn)錄出多個(gè)lncRNAs, 提示HOTAIR可能只是這一個(gè)調(diào)控現(xiàn)象的其中一個(gè)已知的lncRNA，可能還有多個(gè)lncRNAs發(fā)揮類似的作用58。MALAT1 RNA是一個(gè)位于人類1lql3染色體上，長(zhǎng)約8600bp的lncRNA，它可通過(guò)與其他分子(如轉(zhuǎn)錄因子等)相結(jié)合而發(fā)揮基因調(diào)控作用。Lin等119研究發(fā)現(xiàn)MALAT1與肝癌的發(fā)生、發(fā)展密切相關(guān)。在肝細(xì)胞型肝癌中其表達(dá)量是正常

8、肝臟的6倍，同時(shí)該轉(zhuǎn)錄子在肝細(xì)胞型肝癌發(fā)展的各個(gè)階段，包括早期階段，均有顯著的表達(dá)增加，而且在其他非肝細(xì)胞型肝癌中也有類似的差異表達(dá)。通過(guò)原位雜交技術(shù)又發(fā)現(xiàn)在5080的乳腺癌、胰腺癌、結(jié)腸癌以及25的前列腺癌組織中均有MALAT1的高表達(dá)，而在對(duì)照的正常組織中卻僅有低表達(dá)或沒(méi)有表達(dá)。另外，Ji等120發(fā)現(xiàn)相對(duì)于非轉(zhuǎn)移性的非小細(xì)胞肺癌，轉(zhuǎn)移性非小細(xì)胞肺癌中MALATl的表達(dá)更多。Yamada等121報(bào)道在子宮內(nèi)膜間質(zhì)肉瘤中有比正常的子宮內(nèi)膜更多的MALAT1的表達(dá)。因而，MALAT1可能是一個(gè)潛在的廣譜的人類腫瘤標(biāo)志物。通過(guò)激活p53介導(dǎo)腫瘤抑制作用MEG3是最早報(bào)道的具有腫瘤抑制作用的lncR

9、NA。MEG3基因表達(dá)與人類多種正常組織中，而在腦和垂體組織中表達(dá)最高156,157。MEG3在腦腫瘤及大多數(shù)腫瘤細(xì)胞系中均不能檢出，提示其具有腫瘤抑制作用。更重要的是，過(guò)表達(dá)MEG3 RNA能抑制多種腫瘤細(xì)胞系的增殖，進(jìn)一步支持MEG3的腫瘤抑制作用157。在非功能性的垂體瘤中，MEG3調(diào)控區(qū)域的超甲基化與MEG3的失活有關(guān)，為MEG3的失活提供了直接證據(jù)157。功能上，MEG3是有關(guān)高水平的調(diào)控性RNA，因?yàn)樗芗せ頟53依賴性和非依賴性途徑。MEG3介導(dǎo)的p53激活依賴于MEG3 RNA的二級(jí)結(jié)構(gòu)而不是一級(jí)結(jié)構(gòu)158。相反，新發(fā)現(xiàn)的lncRNA-p21被認(rèn)為是p53轉(zhuǎn)錄反應(yīng)的下游抑制子，

10、提示p53轉(zhuǎn)錄網(wǎng)絡(luò)包含了多個(gè)lncRNAs164。通過(guò)誘餌或miRNA海綿作用清除miRNA拷貝數(shù)改變和轉(zhuǎn)錄后沉默等機(jī)制會(huì)導(dǎo)致腫瘤miRNA表達(dá)的異常165-168。兩個(gè)新發(fā)現(xiàn)的lncRNAs被認(rèn)為能通過(guò)自然miRNA海綿（naturalmiRNA sponges）作用降低miRNA表達(dá)水平。HULC基因位于6p24.3染色體上,其編碼的lncRNA具有典型的mRNA特點(diǎn)。盡管HULC與核糖體共同純化，卻沒(méi)有任何翻譯產(chǎn)物，因此它屬于非編碼轉(zhuǎn)錄子170。除了肝癌以外，HULC在結(jié)直腸癌肝臟轉(zhuǎn)移瘤和產(chǎn)乙型肝炎病毒的肝細(xì)胞癌細(xì)胞系均高表達(dá)171。最近研究初步揭示肝癌細(xì)胞HULC高表達(dá)及其作用的機(jī)制1

11、72。HULC是復(fù)雜的自主調(diào)控網(wǎng)絡(luò)的一部分，該網(wǎng)絡(luò)調(diào)控異常會(huì)導(dǎo)致HULC表達(dá)的上調(diào)HULC RNA可能是通過(guò)分子誘餌（molecular decoy）或miRNA海綿（miRNA sponge）作用清除miR-372。miR-372能轉(zhuǎn)錄抑制作用于cAMP反應(yīng)元件結(jié)合蛋白（CREB）的一種激酶PRKACB。CREB一旦激活，CREB能通過(guò)維持HULC啟動(dòng)子區(qū)域的一個(gè)開放讀碼框架促進(jìn)HULC的轉(zhuǎn)錄172。假基因?qū)Γ≒seudogene pairs）PTEN和PTENP1抑癌基因PTEN及其相應(yīng)的lncRNAPTEN假基因1（PTENP1）的相互作用機(jī)制類似于HULC RNA 173。PTEN/P

12、TENP1的轉(zhuǎn)錄子具有類似的3非翻譯區(qū)（UTRs），使兩者均結(jié)合于相同的miRNAs。通過(guò)結(jié)合于miRNAs，PTENP1能減少miRNAs對(duì)PTEN的抑制作用而上調(diào)其表達(dá)。在癌癥中，特異性突變使PTENP1的miRNA結(jié)合位點(diǎn)失活將減少抑癌基因PTEN的轉(zhuǎn)錄，從而促進(jìn)腫瘤細(xì)胞的增殖173。這種機(jī)制有重要的意義，因?yàn)镻TEN1水平的輕微改變即可影響癌癥的易感性173。介導(dǎo)腫瘤抑制因子的異常表達(dá)ANRIL基因位于人類9p21.3染色體上，是42kb的INK4b-ARF-INK4a位點(diǎn)的一部分。ANRIL是由RNA多聚酶II 轉(zhuǎn)錄，并被剪切成不同的異型體，包括一個(gè)名為p15AS的長(zhǎng)34.8kb的非

13、剪切轉(zhuǎn)錄子175,176。ANRIL的異常表達(dá)及單核苷酸多態(tài)性與包括癌癥在內(nèi)的多種疾病發(fā)生的易感性關(guān)系密切181,182。在白血病、黑色素瘤、肺癌和膀胱癌中，INK4b-ARF-INK4a位點(diǎn)容易發(fā)生高頻突變和超甲基化181。 研究顯示ANRIL能夠轉(zhuǎn)錄后沉默抑癌基因p15，盡管其機(jī)制尚未完全清楚175。癌癥類型特異性表達(dá)盡管上述大部分lncRNAs均在多種人類組織中表達(dá)，但有極少數(shù)lncRNAs到目前為止只發(fā)現(xiàn)表達(dá)于某種組織中。例如HOTAIR只在乳腺癌表達(dá)，而lncRNAs PCGEM1,DD3和PCNCR1僅與前列腺癌有關(guān)68,186,187。其次，肝臟相關(guān)性lncRNAsHULC僅高表

14、達(dá)于原發(fā)性肝細(xì)胞癌和結(jié)直腸癌肝轉(zhuǎn)移瘤中，而不在原發(fā)性結(jié)直腸癌或非肝轉(zhuǎn)移瘤表達(dá)171。RNA多聚酶III轉(zhuǎn)錄到目前為止，上述lncRNAs均由RNA多聚酶II轉(zhuǎn)錄,但仍然有很多ncRNAs是由RNA 多聚酶III（RNA pol III）轉(zhuǎn)錄191。重要的是RNA pol III在癌癥細(xì)胞常常異常調(diào)控而導(dǎo)致其活性的增加192,193。促使腫瘤細(xì)胞中RNA pol III活性增加的機(jī)制包括RNA pol III轉(zhuǎn)錄因子的高表達(dá)，RNA pol III抑制子逃避和直接的原癌基因激活193-195。RNA pol III功能異常會(huì)影響由此多聚酶轉(zhuǎn)錄的lncRNAs的表達(dá)。例如，BC200是靈長(zhǎng)類神經(jīng)系

15、統(tǒng)神經(jīng)元及人類非神經(jīng)元癌癥細(xì)胞的胞漿內(nèi)的lncRNA20,50,51,196,197。與上面我們提到的lncRNAs不同的是，BC200是由RNA pol III所轉(zhuǎn)錄，并與人類Alu元件有獨(dú)特的同源性。BC200是eIF4A依賴性翻譯啟動(dòng)的負(fù)性調(diào)控子199。由于大部分全轉(zhuǎn)錄組測(cè)序均應(yīng)用富集多聚A純化轉(zhuǎn)錄子的方法，RNA pol III轉(zhuǎn)錄子可能會(huì)從這些分析中排除。這提示，其他尚未確定的RNA pol III轉(zhuǎn)錄的lncRNAs在惡性腫瘤中的高表達(dá)可能參與了腫瘤的發(fā)生。二、 lncRNA在癌癥診斷和治療的應(yīng)用與非編碼的miRNA一樣，lncRNAs 是具有廣闊的疾病診斷和治療應(yīng)用前景。在某種癌

16、癥中特異性表達(dá)的lncRNAs 的差異表達(dá)或高表達(dá)可用以開發(fā)新型的腫瘤標(biāo)志物，這些lncRNAs 可能與病人對(duì)化療的敏感性密切相關(guān)。加深對(duì)lncRNAs 作用機(jī)制的理解將提供調(diào)控基因表達(dá)的新方法，例如開發(fā)與miRNA結(jié)合位點(diǎn)競(jìng)爭(zhēng)的類似物，染色質(zhì)重塑因子或DNA。研究者指出介導(dǎo)轉(zhuǎn)錄基因沉默（transcriptional gene silencing， TGS）途徑，尤其是抑癌基因和原癌基因TGS途徑有可能為開發(fā)新型治療方法的提供新的思路205。三、 結(jié)語(yǔ)最近的研究表明，一些長(zhǎng)片段ucRNA的高表達(dá)或低表達(dá)與某些腫瘤的發(fā)生、發(fā)展有關(guān)。然而研究尚處于初級(jí)階段，利用長(zhǎng)片段ncRNA進(jìn)行腫瘤的診斷和治

17、療還有許多尚未解決的問(wèn)題。在診斷方面，如何確定不同臨床分期的特征性長(zhǎng)片段ncRNA譜，以達(dá)到早期診斷的目的；如何確定不同病理類型的特征性長(zhǎng)片段ncRNA譜，以達(dá)到病理分型的目的；如何尋找無(wú)創(chuàng)手段進(jìn)行檢測(cè)等。在治療方面，如何設(shè)計(jì)持續(xù)高效表達(dá)的載體；如何有效導(dǎo)入至活體內(nèi)；如何在特定組織或器官持續(xù)表達(dá)等都是亟待解決的問(wèn)題。相信隨著長(zhǎng)片段ncRNA研究技術(shù)的發(fā)展，在不久的將來(lái)長(zhǎng)片段ncRNA會(huì)為腫瘤的診治帶來(lái)突破性的進(jìn)展。References1. Stein LD: Human genome: end of the beginning. Nature 2004,431(7011):915-916.2.

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