陳青 代智 周儉

（復(fù)旦大學(xué)附屬中山醫(yī)院肝癌研究所 上海 200032）

腫瘤的發(fā)生、發(fā)展與表觀遺傳異常改變密切相關(guān)。5-胞嘧啶堿基（5-position of cytosine，5-mC）甲基化和RNA的N6-甲基腺苷（N6-methyladenosine，m-6-A）是常見并極其重要的表觀修飾，具有高度保守性，存在于所有高等真核生物體中[1-3]，參與維持基因組的穩(wěn)定和表達(dá)，并在機(jī)體生長發(fā)育過程中起重要作用[4-5]。DNA甲基化修飾調(diào)控正常生長發(fā)育過程，包括X染色體失活、印記和轉(zhuǎn)錄調(diào)節(jié)。異常DNA甲基化是腫瘤的重要的表觀特征，參與腫瘤的發(fā)生、發(fā)展[6-8]，啟動子區(qū)域甲基化修飾可導(dǎo)致轉(zhuǎn)錄抑制。研究表明發(fā)現(xiàn)TET（ten-eleven translocation）酶家族包括TET1、2、3，可催化5-mC成為5-hmC[9-10]。RNA的m-6-A修飾也是常見的表觀修飾，類似于DNA的5-mC修飾。研究表明RNA的m-6-A表觀修飾可影響RNA的轉(zhuǎn)錄、代謝、剪接、穩(wěn)定性和蛋白與其結(jié)合等，參與精子形成、發(fā)育等重要的生物學(xué)過程[11-12]。研究發(fā)現(xiàn)，RNA脫甲基酶肥胖風(fēng)險(xiǎn)基因（fat mass and obesity-associated，F(xiàn)TO）和 ALKBH5可擦去RNA的m-6-A印記。本文綜述DNA與RNA甲基化表觀修飾的生物學(xué)特征及其在腫瘤中的作用。

1 5-hmC

1.1 5-hmC的主要生物學(xué)功能

1952年，人類在噬菌體DNA中發(fā)現(xiàn)5-hmC的存在。由于受技術(shù)限制，5-hmC研究一直未得到重視。初步研究發(fā)現(xiàn)5-hmC參與了DNA去甲基化過程，能降低MeCP蛋白的甲基化結(jié)合結(jié)構(gòu)域（MBD）與甲基化DNA的親和性，具有潛在的參與基因表達(dá)調(diào)控和轉(zhuǎn)錄調(diào)節(jié)功能。5-hmC亦可能參與基因表達(dá)調(diào)控過程，已成為當(dāng)前表觀遺傳學(xué)領(lǐng)域的研究熱點(diǎn)之一。最近發(fā)現(xiàn)TET蛋白酶家族（TET1、2、3）通過催化5-mC產(chǎn)生5-hmC，逆轉(zhuǎn)甲基化狀態(tài)，引起人們對5-hmC的研究興趣。

5-mC被稱為“第5堿基”，長期認(rèn)為是位于基因組DNA唯一的表觀遺傳共價修飾。5-mC常見于哺乳動物CpG島的二核苷酸，除此以外，非CpG甲基化只見于多能干細(xì)胞中[13-14]。CpG島甲基化在基因轉(zhuǎn)錄沉默、RNA剪接、DNA修復(fù)、基因組印記和X染色體失活中有重要作用，參與分化、發(fā)育和疾病過程，包括惡性腫瘤[15-18]。研究發(fā)現(xiàn)在DNA甲基轉(zhuǎn)移酶家族（DNMT）作用下，以S-腺苷甲硫氨酸（SAM）提供甲基供體，將其轉(zhuǎn)移到脫氧胞嘧啶環(huán)第5位碳原子上形成甲基化脫氧胞嘧啶，產(chǎn)生5-mC。哺乳動物的甲基轉(zhuǎn)移酶家族有5個成員（DNMT1、DNMT2、DNMT3A、DNMT 3B和DNMT 3L），其中只有DNMT1、DNMT3A和DNMT3B具有甲基轉(zhuǎn)移活性。DNMT1參與維持甲基化狀態(tài)，DNMT2可與DNA上特異位點(diǎn)結(jié)合，但具體作用尚不清楚。甲基化轉(zhuǎn)移酶DNMT3A和DNMT3B高表達(dá)于胚胎干細(xì)胞（ESCs），參與哺乳動物胚胎發(fā)育和細(xì)胞分化過程中基因DNA甲基化的建立。DNMT3B是胚胎早期發(fā)育所必須，而DNMT3A是胚胎發(fā)育晚期所必須；DNMT3L可增強(qiáng)DNMT3A酶活性，本身并無催化活性[19]。研究發(fā)現(xiàn)5-mC在人類細(xì)胞分化、發(fā)育和腫瘤疾病過程中起著極其重要的調(diào)節(jié)作用，特別是在CpG島甲基化所致抑癌基因轉(zhuǎn)錄失活中。

1.2 5-hmC調(diào)控基因表達(dá)

5-hmC被稱為“第6個堿基”，在2-酮戊二酸（α-ketoglutarate，α-KG）和Fe2+作用下，雙加氧酶TET酶家族TET1、2、3可將5-mC羥化生成5-hmC[18-19]。最近研究發(fā)現(xiàn)，在人、小鼠大腦及胚胎干細(xì)胞中5-hmC高表達(dá)，常位于轉(zhuǎn)錄起始點(diǎn)[18-19]。研究提示，5-hmC特異性在基因體部及啟動子區(qū)域富集，深入研究發(fā)現(xiàn)基因轉(zhuǎn)錄起始點(diǎn)和基因體部的5-hmC水平與基因表達(dá)呈正相關(guān)[20-21]。然而，Xu等[22]發(fā)現(xiàn)看家基因體部的5-hmC水平仍然很低，基因體部的5-hmC水平與基因表達(dá)高低也不是簡單的線性關(guān)系。此外，Stroud等[23]研究發(fā)現(xiàn)，5-hmC與組蛋白H3K4me1和H3K27ac修飾呈正相關(guān)，同時增強(qiáng)子區(qū)域的5-hmC水平反映增強(qiáng)子處于激活或靜息狀態(tài)。因此，5-hmC具有潛在的參與基因表達(dá)調(diào)控的功能。

1.3 5-hmC在腫瘤中表達(dá)缺失

大量研究表明，實(shí)體腫瘤中5-hmC缺失，如黑色素瘤、乳腺癌、肝癌、前列腺癌、肺癌和胰腺癌等[6,24-25]。文獻(xiàn)報(bào)道腸癌組織中5-hmC含量顯著下降，甚至結(jié)腸癌細(xì)胞中幾乎無法檢測到5-hmC表達(dá)[26]。Kudo等[27]通過免疫印跡法研究肝癌、腸癌、肺癌等組織與對應(yīng)的正常組織中5-hmC水平，發(fā)現(xiàn)腫瘤組織中5-hmC水平顯著降低。此外，文獻(xiàn)報(bào)道腦膠質(zhì)瘤常見5-hmC缺失，且5-hmC缺失與腫瘤患者不良預(yù)后密切相關(guān)[28-29]。Lian等[6]研究發(fā)現(xiàn)，黑色素瘤中5-hmC水平顯著降低，且5-hmC缺失與腫瘤細(xì)胞分化密切相關(guān)；TET2和異檸檬酸脫氫酶（isocitrate dehydrogenase，IDH）2表達(dá)下調(diào)可導(dǎo)致5-hmC缺失，重新導(dǎo)入活性TET2和IDH2，重建黑色素瘤細(xì)胞中5-hmC，能顯著抑制黑色素瘤生長[28]。由此推測，5-hmC參與黑色素瘤的發(fā)展，IDH2和TET蛋白下調(diào)是5-hmC水平降低的機(jī)制之一。此外，大量文獻(xiàn)報(bào)道血液系統(tǒng)惡性腫瘤亦常伴有5-hmC缺失[29-32]。

1.4 腫瘤中5-hmC缺失的機(jī)制

在α-KG和Fe2+作用下，TET1、2、3可將5-mC催化生成5-hmC。研究發(fā)現(xiàn)，腫瘤中常見TET酶表達(dá)異常，影響其介導(dǎo)DNA去甲基化，導(dǎo)致腫瘤發(fā)生、發(fā)展[32]。此外，野生型的IDH（包括IDH1和IDH2）可催化異檸檬酸，產(chǎn)生a-KG，然而突變型IDH1、IDH2催化異檸檬酸產(chǎn)生 2-羥戊二酸（2-hydroxyglutarate，2-HG），而 2-HG是一種在結(jié)構(gòu)上類似α-KG的分子，能拮抗α-KG，競爭性抑制多種α-KG依賴性的雙加氧酶TET蛋白酶家族[6,29,33-35]。惡性腫瘤常見IDH1和IDH2編碼基因高頻突變，不僅失去正常的酶催化活性，還因產(chǎn)生2-HG而降低了α-KG的產(chǎn)量[36]。因此，腫瘤組織中的TET1、2、3和IDH1、IDH2酶轉(zhuǎn)錄水平下調(diào)可能是導(dǎo)致5-hmC缺失的原因。此外，文獻(xiàn)報(bào)道m(xù)iR-22通過拮抗TET酶介導(dǎo)5-hmC缺失，從而促進(jìn)乳腺癌侵襲轉(zhuǎn)移，還可導(dǎo)致miR-200啟動子5-hmC缺失，影響miR-200表達(dá)，miR-200可負(fù)性調(diào)控腫瘤細(xì)胞上皮細(xì)胞間質(zhì)轉(zhuǎn)化（epithelial-tomesenchymal transition，EMT）[37]。研究發(fā)現(xiàn)，維生素C可影響胚胎干細(xì)胞和體細(xì)胞基因組重排，在TET酶去甲基化過程中發(fā)揮重要作用[38-39]?？傊?，大量文獻(xiàn)證實(shí)，5-hmC可能對惡性腫瘤形成和發(fā)生、發(fā)展具有負(fù)性調(diào)控作用，可能原因是腫瘤組織中5-hmC缺失，5-mC水平升高，導(dǎo)致基因組處于高甲基化狀態(tài)，大量抑癌基因失活或凋亡相關(guān)基因未被激活，促進(jìn)癌細(xì)胞增殖，免于被凋亡或受抑癌基因調(diào)控。5-hmC在腫瘤的發(fā)生、發(fā)展中的具體機(jī)制仍不清楚，需要進(jìn)一步深入研究。

2 m-6-A

2.1 m-6-A的生物學(xué)功能

1974年，科學(xué)家首次發(fā)現(xiàn)m-6-A，當(dāng)時不能確定這一發(fā)現(xiàn)是否是其他RNA分子污染的結(jié)果[40]。RNA的m-6-A修飾常被稱為mRNA的第5種堿基，幾乎存在于所有高等真核生物體中。研究發(fā)現(xiàn)m-6-A常位于mRNA的終止密碼子附近，在多種脊椎動物mRNA的高度保守域，是經(jīng)過數(shù)億年進(jìn)化選擇保存下來的，m-6-A修飾對人類及其他動物都至關(guān)重要[41]。另外，研究發(fā)現(xiàn)長鏈非編碼RNA存在m-6-A修飾，影響其穩(wěn)定性和代謝[41-42]。RNA的m-6-A修飾的發(fā)現(xiàn)成為開辟RNA甲基化研究新的途徑，m-6-A修飾作為RNA新一層次的調(diào)控，被稱為RNA表觀遺傳[43-44]。

文獻(xiàn)報(bào)道約20%的人類mRNA可被常規(guī)的甲基化修飾，約7 000多種不同的mRNA分子有m-6-A修飾[42]，這意味著m-6-A修飾可能廣泛地影響著基因表達(dá)。高通量測序發(fā)現(xiàn)m-6-A修飾主要位于mRNAs外顯子區(qū)域和3’-非編碼區(qū)域（3’-UTR）[12]。因此，RNA的m-6-A修飾有可能影響miRNA與靶基因的mRNA互補(bǔ)結(jié)合序列。研究發(fā)現(xiàn)RNA的m-6-A修飾可改變RNA結(jié)構(gòu)通過弱化堿基配對，此外RNA的m-6-A修飾可增加蛋白質(zhì)結(jié)合識別位點(diǎn)，綁定蛋白招募其他蛋白復(fù)合物參與細(xì)胞生物學(xué)過程，包括mRNA剪接、RNA輸出、穩(wěn)定性和免疫耐受[45]。

2.2 影響m-6-A表達(dá)的因素

甲基轉(zhuǎn)移酶METTL3參與維持人類RNA的m-6-A[46-47]。METTL3參與調(diào)控細(xì)胞生存和發(fā)育的相關(guān)信號通路[48]。研究發(fā)現(xiàn)RNA干擾敲除甲基轉(zhuǎn)移酶METTL3，可增加細(xì)胞凋亡并促進(jìn)其死亡[49]，提示m-6-A修飾對細(xì)胞生存起著極其重要的調(diào)控作用。此外，甲基轉(zhuǎn)移酶METTL14與METTL3形成的甲基轉(zhuǎn)移酶復(fù)合物會影響m-6-A表達(dá)，參與調(diào)節(jié)RNA代謝[50]。

研究人員鑒定mRNA去甲基化酶，發(fā)現(xiàn)FTO可將mRNA中m-6-A擦除，逆轉(zhuǎn)到常規(guī)腺苷[43-44]。目前，關(guān)于FTO研究較多的是其突變導(dǎo)致肥胖和糖尿病[51-54]。據(jù)估計(jì)，全球10億人有FTO突變，此突變是肥胖癥及2型糖尿病的主要病因。此外，另一種去甲基化酶ALKBH5，類似于FTO，是ALKB同源基因家族，可逆轉(zhuǎn)RNA的m-6-A修飾，參與RNA的輸出和代謝，文獻(xiàn)報(bào)道ALKBH5缺失損害老鼠的生殖功能[55]。

2.3 腫瘤中m-6-A異常表達(dá)

RNA作為連接基因組和蛋白組的橋梁，RNA的m-6-A修飾是可逆修飾，在維持機(jī)體生理狀態(tài)及某些病理過程中發(fā)揮著重要作用。文獻(xiàn)報(bào)道m(xù)-6-A出現(xiàn)在許多人類疾病基因編碼的mRNA中，包括大腦疾?。ㄈ绻陋?dú)癥、阿爾茨海默病、精神分裂癥）、糖尿病、肥胖以及癌癥[56-57]。mRNA非常復(fù)雜，RNA甲基化異?？梢鸲喾N疾病。目前，關(guān)于RNA的m-6-A修飾在腫瘤發(fā)生、發(fā)展中的作用鮮見報(bào)道。僅有少量報(bào)道提示FTO與乳腺癌等腫瘤發(fā)生、發(fā)展關(guān)系密切，但具體機(jī)制尚未闡明[58-60]。

3 展望

隨著表觀遺傳學(xué)的研究深入及高通量測序技術(shù)的發(fā)展，人類有望揭開5-hmC和m-6-A表觀改變在生命活動過程和疾?。ò◥盒阅[瘤）過程中的重要作用。深入研究5-hmC和m-6-A表觀遺傳異常改變的去甲基化酶，包括DNA去甲基化酶TET酶家族和RNA去甲基化酶FTO和ALKBH5，參與腫瘤發(fā)生、發(fā)展和轉(zhuǎn)移復(fù)發(fā)的分子機(jī)制很有必要。因此，應(yīng)用修復(fù)異常表觀遺傳改變的藥物，將來有可能逆轉(zhuǎn)惡性分化的腫瘤細(xì)胞，成功治愈或改造腫瘤患者。

[1] Law JA, Jacobsen SE. Establishing, maintaining and modifying DNA methylation patterns in plants and animals[J].Nat Rev Genet, 2010, 11(3): 204-220.

[2] Tuck MT. The formation of internal 6-methyladenine residues in eucaryotic messenger RNA[J]. Int J Biochem, 1992, 24(3):379-386.

[3] Jia G, Fu Y, He C. Reversible RNA adenosine methylation in biological regulation[J]. Trends Genet, 2013, 29(2): 108-115.

[4] Jaenisch R, Bird A. Epigenetic regulation of gene expression:how the genome integrates intrinsic and environmental signals[J]. Nat Genet, 2003, 33(Suppl): 245-254.

[5] Smith ZD, Meissner A. DNA methylation: roles in mammalian development[J]. Nat Rev Genet, 2013, 14(3):204-220.

[6] L i a n C G, X u Y, C e o l C, e t a l. L o s s o f 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma[J]. Cell, 2012, 150(6): 1135-1146.

[7] Baylin SB, Jones PA. A decade of exploring the cancer epigenome-biological and translational implications[J]. Nat Rev Cancer, 2011, 11(10): 726-734.

[8] Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps[J]. Nat Rev Genet, 2007, 8(4):286-298.

[9] Ito S, D’Alessio AC, Taranova OV, et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification[J]. Nature, 2010, 466(7310): 1129-1133.

[10] Zhang H, Zhang X, Clark E, et al. TET1 is a DNA-binding protein that modulates DNA methylation and gene transcription via hydroxylation of 5-methylcytosine[J]. Cell Res, 2010, 20(12): 1390-1393.

[11] Wang X, Lu Z, Gomez A, et al. N6-methyladenosinedependent regulation of messenger RNA stability[J]. Nature,2014, 505(7481): 117-120.

[12] Niu Y, Zhao X, Wu YS, et al. N6-methyl-adenosine (m6A) in RNA: an old modification with a novel epigenetic function[J].Genomics Proteomics Bioinformatics, 2013, 11(1): 8-17.

[13] Ramsahoye BH, Biniszkiewicz D, Lyko F, et al. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a[J]. Proc Natl Acad Sci U S A, 2000, 97(10): 5237-5242.

[14] Lister R, Pelizzola M, Dowen RH, et al. Human DNA methylomes at base resolution show widespread epigenomic differences[J]. Nature, 2009, 462(7271): 315-322.

[15] Bird A. DNA methylation patterns and epigenetic memory[J].Genes Dev, 2002, 16(1): 6-21.

[16] Ndlovu MN, Denis H, Fuks F. Exposing the DNA methylome iceberg[J]. Trends Biochem Sci, 2011, 36(7): 381-387.

[17] Laurent L, Wong E, Li G, et al. Dynamic changes in the human methylome during differentiation[J]. Genome Res,2010, 20(3): 320-331.

[18] Guo JU, Su Y, Zhong C, et al. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain[J]. Cell, 2011, 145(3): 423-434.

[19] Auclair G, Weber M. Mechanisms of DNA methylation and demethylation in mammals[J]. Biochimie, 2012, 94(11):2202-2211.

[20] Jin SG, Wu X, Li AX, et al. Genomic mapping of 5-hydroxymethylcytosine in the human brain[J]. Nucleic Acids Res, 2011, 39(12): 5015-5024.

[21] Song CX, Szulwach KE, Fu Y, et al. Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine[J]. Nat Biotechnol, 2011, 29(1): 68-72.

[22] Xu Y, Wu F, Tan L, et al. Genome-wide regulation of 5hmC,5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells[J]. Mol Cell, 2011, 42(4): 451-464.

[23] Stroud H, Feng S, Morey Kinney S, et al.5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells[J]. Genome Biol,2011, 12(6): R54.

[24] Yang H, Liu Y, Bai F, et al. Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation[J]. Oncogene, 2013, 32(5): 663-669.

[25] Hsu CH, Peng KL, Kang ML, et al. TET1 suppresses cancer invasion by activating the tissue inhibitors of metalloproteinases[J]. Cell Rep, 2012, 2(3): 568-579.

[26] Wen L, Li X, Yan L, et al. Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain[J]. Genome Biol, 2014, 15(3):R49.

[27] Kudo Y, Tateishi K, Yamamoto K, et al. Loss of 5-hydroxymethylcytosine is accompanied with malignant cellular transformation[J]. Cancer Sci, 2012, 103(4): 670-676.

[28] Orr BA, Haffner MC, Nelson WG, et al. Decreased 5-hydroxymethylcytosine is associated with neural progenitor phenotype in normal brain and shorter survival in malignant glioma[J]. PLoS One, 2012, 7(7): e41036.

[29] Xu W, Yang H, Liu Y, et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases[J]. Cancer Cell,2011, 19(1): 17-30.

[30] Song SJ, Ito K, Ala U, et al. The oncogenic microRNA miR-22 targets the TET2 tumor suppressor to promote hematopoietic stem cell self-renewal and transformation[J].Cell Stem Cell, 2013, 13(1): 87-101.

[31] Huang H, Jiang X, Li Z, et al. TET1 plays an essential oncogenic role in MLL-rearranged leukemia[J]. Proc Natl Acad Sci U S A, 2013, 110(29): 11994-11999.

[32] Ko M, Huang Y, Jankowska AM, et al. Impairedhydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2[J]. Nature, 2010, 468(7325): 839-843.

[33] Figueroa ME, Abdel-Wahab O, Lu C, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impairhematopoietic differentiation[J]. Cancer Cell, 2010, 18(6): 553-567.

[34] Delhommeau F, Dupont S, Della Valle V, et al. Mutation in TET2 in myeloid cancers[J]. N Engl J Med, 2009, 360(22):2289-2301.

[35] Mullighan CG. TET2 mutations in myelodysplasia and myeloid malignancies[J]. Nat Genet, 2009, 41(7): 766-767.

[36] Parsons DW, Jones S, Zhang X, et al. An integrated genomic analysis of human glioblastoma multiforme[J]. Science, 2008,321(5897): 1807-1812.

[37] Song SJ, Poliseno L, Song MS, et al. MicroRNA-antagonism regulates breast cancer stemness and metastasis via TET-family-dependent chromatin remodeling[J]. Cell, 2013,154(2): 311-324.

[38] Blaschke K, Ebata KT, Karimi MM, et al. Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells[J]. Nature, 2013, 500(7461): 222-226.

[39] Chen J, Guo L, Zhang L, et al. Vitamin C modulates TET1 function during somatic cell reprogramming[J]. Nat Genet,2013, 45(12): 1504-1509.

[40] Desrosiers R, Friderici K, Rottman F. Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells[J]. Proc Natl Acad Sci U S A, 1974, 71(10):3971-3975.

[41] Meyer KD, Saletore Y, Zumbo P, et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3’ UTRs and near stop codons[J]. Cell, 2012, 149(7): 1635-1646.

[42] Dominissini D, Moshitch-Moshkovitz S, Schwartz S, et al.Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq[J]. Nature, 2012, 485(7397): 201-206.

[43] He C. Grand challenge commentary: RNA epigenetics?[J].Nat Chem Biol, 2010, 6(12): 863-865.

[44] Yi C, Pan T. Cellular dynamics of RNA modification[J]. Acc Chem Res, 2011, 44(12): 1380-1388.

[45] Pan T. N6-methyl-adenosine modification in messenger and long non-coding RNA[J]. Trends Biochem Sci, 2013, 38(4):204-209.

[46] Bokar JA, Shambaugh ME, Polayes D, et al. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase[J]. RNA, 1997,3(11): 1233-1247.

[47] Bokar JA, Rath-Shambaugh ME, Ludwiczak R, et al.Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex[J]. J Biol Chem, 1994, 269(26): 17697-17704.

[48] Hongay CF, Orr-Weaver TL. Drosophila Inducer of MEiosis 4(IME4) is required for Notch signaling during oogenesis[J].Proc Natl Acad Sci U S A, 2011, 108(36): 14855-14860.

[49] Lavi U, Fernandez-Mu?oz R, Darnell JE, Jr. Content of N-6 methyl adenylic acid in heterogeneous nuclear and messenger RNA of HeLa cells[J]. Nucleic Acids Res, 1977, 4(1): 63-69.

[50] Ping XL, Sun BF, Wang L, et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase[J]. Cell Res, 2014, 24(2): 177-189.

[51] Benedict C, Axelsson T, S?derberg S, et al. Fat mass and obesity-associated gene(FTO) is linked to higher plasma levels of the hunger hormone ghrelin andlower serum levels of the satiety hormone leptin in older adults[J]. Diabetes,2014, 63(11): 3955-3959.

[52] Church C, Moir L, McMurray F, et al. Overexpression of Fto leads to increased food intake and results in obesity[J]. Nat Genet, 2010, 42(12): 1086-1092.

[53] Fischer J, Koch L, Emmerling C, et al. Inactivation of the Fto gene protects from obesity[J]. Nature, 2009, 458(7240): 894-898.

[54] Dina C, Meyre D, Gallina S, et al. Variation in FTO contributes to childhood obesity and severe adult obesity[J].Nat Genet, 2007, 39(6): 724-726.

[55] Zheng G, Dahl JA, Niu Y, et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility[J]. Mol Cell, 2013, 49(1): 18-29.

[56] Chandola U, Das R, Panda B. Role of the N6-methyladenosine RNA mark in gene regulation and its implications on development and disease[J]. Brief Funct Genomics, 2014 Oct 10. pii: elu039. [Epub ahead of print].

[57] Klungland A, Dahl JA. Dynamic RNA modifications in disease[J]. Curr Opin Genet Dev, 2014 Jun; 26:47-52. doi:10.1016/j.gde.2014.05.006. Epub 2014 Jul 5.

[58] Li G, Chen Q, Wang L, et al. Association between FTO gene polymorphism and cancer risk: evidence from 16 277 cases and 31 153 controls[J]. Tumour Biol, 2012, 33(4): 1237-1243.

[59] Garcia-Closas M, Couch FJ, Lindstrom S, et al. Genome-wide association studies identify four ER negative-specific breast cancer risk loci[J]. Nat Genet, 2013, 45(4): 392-398, 398e1-2.

[60] Hernández-Caballero ME, Sierra-Ramírez JA. Single nucleotide polymorphisms of the FTO gene and cancer risk:an overview[J]. Mol Biol Rep, 2015, 42(3): 699-704.