m6A 甲基化研究方法

./RNA甲基化修饰〔m6A〕研究思路与方案设计RNA甲基化修饰约占所有RNA修饰的60%以上,而N6-甲基腺嘌呤〔N6-methyladenosine,m6A〕是高等生物mRNA和lncRNAs上最为普遍的修饰。目前发现microRNA,circRNA,rRNA,tRNA和snoRNA上都有发生m6A修饰。m6A修饰主要发生在RRACH序列中的腺嘌呤上,其功能由"编码器<Writer>"、"消码器<Eraser>"和"读码器<Reader>"决定ADDINEN.CITEADDINEN.CITE.DATA[\o"Fu,2014#8522"1]。"编码器<Writer>"即甲基转移酶,目前已知这个复合物的成分有METTL3,METTL14,WTAP和KIAA1429；而ALKBH5和FTO作为去甲基酶〔消码器〕可逆转甲基化；m6A由m6A结合蛋白识别,目前发现m6A结合蛋白〔读码器〕有YTH结构域蛋白〔包括YTHDF1,YTHDF2,YTHDF3,YTHDC1和YTHDC2〕和核不均一蛋白HNRNP家族〔HNRNPA2B1和HNRNPC〕。m6A酶系统METTL3是早先被鉴定为结合SAM的组件,其缺失引起小鼠胚胎干细胞、Hela细胞和HepG2细胞中m6Apeaks的减少。METTL3与其同源蛋白METTL14定位在富含剪切因子的细胞核内亚细胞器－核小斑〔Nuclearspeckle〕上,显示ｍ６Ａ修饰可能和ＲＮＡ的剪切加工相关。WTAP与METTL3–METTL14二聚体相互作用,并共定位于核小斑,影响甲基化效率,参与mRNA剪。而KIAA1429作为候选的甲基转移酶复合体的新亚基,是整体甲基化进程所必须的ADDINEN.CITEADDINEN.CITE.DATA[\o"Schwartz,2014#8496"2]。FTO是ALKB家族的成员,作为第一个被发现的去甲基酶,可影响剪切因子SRSF2的RNA结合能力,进而调控pre-mRNA的剪切加工过程ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhao,2014#8497"3]。目前已发现FTO调节异常与肥胖、大脑畸形和生长迟缓相关,揭示m6A可能对这些疾病具有重要的调节功能ADDINEN.CITEADDINEN.CITE.DATA[\o"Fu,2013#8498"4-6]。ALKBH5是ALKB家族中被发现具有去甲基作用的另一个成员,以RNaseA敏感的方式与核小斑共定位,它可直接催化m6A-甲基化腺苷去除甲基而不同于FTO的氧化去甲基化ADDINEN.CITEADDINEN.CITE.DATA[\o"Zheng,2013#8501"7].此外,ALKBH5和它的去甲基化活性影响新生mRNA合的成和剪切效率ADDINEN.CITEADDINEN.CITE.DATA[\o"Zheng,2013#8501"7],且ALKBH5敲除雄性小鼠表现出精子发生异常,这可能是精子发生相关基因表达改变的结果ADDINEN.CITEADDINEN.CITE.DATA[\o"Zheng,2013#8501"7]。m6AmRNA修饰执行其功能主要通过两个途径：精细调控甲基化转录本的结构,以阻止或诱使蛋白-RNA相互作用；或被直接由m6A结合蛋白识别,诱发后续反应。目前一类含有YTH功能结构域的蛋白被鉴定为m6A修饰的结合蛋白。其中YTHDF1,YTHDF2,YTHDF3,YTHDC1和YTHDC2己被证实是ｍ６Ａ的结合蛋白.YTHDF1主要影响ｍ６Ａ修饰基因的翻译,YTHDF2主要影响ｍ６Ａ修饰基因的降解,而YTHDC1结合ｍ６Ａ修饰的基因后影响其剪接。HNRNPC是一种丰富的核RNA结合蛋白,参与pre-mRNA的加工ADDINEN.CITE<EndNote><Cite><Author>Cienikova</Author><Year>2014</Year><RecNum>8503</RecNum><DisplayText>[8]</DisplayText><record><rec-number>8503</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8503</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Cienikova,Z.</author><author>Damberger,F.F.</author><author>Hall,J.</author><author>Allain,F.H.</author><author>Maris,C.</author></authors></contributors><auth-address>DepartmentofBiology,InstituteofMolecularBiologyandBiophysics,ETHZurich,8093Zurich,Switzerland.</auth-address><titles><title>Structuralandmechanisticinsightsintopoly<uridine>tractrecognitionbythehnRNPCRNArecognitionmotif</title><secondary-title>JAmChemSoc</secondary-title><alt-title>JournaloftheAmericanChemicalSociety</alt-title></titles><pages>14536-44</pages><volume>136</volume><number>41</number><edition>2014/09/13</edition><keywords><keyword>Heterogeneous-NuclearRibonucleoproteinGroupC/*chemistry</keyword><keyword>Humans</keyword><keyword>Kinetics</keyword><keyword>Models,Molecular</keyword><keyword>MolecularStructure</keyword><keyword>PolyU/*chemistry</keyword><keyword>RNA/*chemistry</keyword><keyword>Thermodynamics</keyword></keywords><dates><year>2014</year><pub-dates><date>Oct15</date></pub-dates></dates><isbn>1520-5126<Electronic> 0002-7863<Linking></isbn><accession-num>25216038</accession-num><work-type>ResearchSupport,Non-U.S.Gov't</work-type><urls><related-urls><url>/pubmed/25216038</url></related-urls></urls><electronic-resource-num>10.1021/ja507690d</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Cienikova,2014#8503"8],且研究表明HNRNPC通过m6A与RNA结合调控目标转录本的丰度和选择性剪切ADDINEN.CITEADDINEN.CITE.DATA[\o"Liu,2015#8504"9].图1m6A修饰的酶系统ADDINEN.CITE<EndNote><Cite><Author>Zhao</Author><Year>2017</Year><RecNum>8521</RecNum><DisplayText>[10]</DisplayText><record><rec-number>8521</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8521</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Zhao,B.S.</author><author>Roundtree,I.A.</author><author>He,C.</author></authors></contributors><auth-address>DepartmentofChemistry,DepartmentofBiochemistryandMolecularBiology,andInstituteforBiophysicalDynamics,HowardHughesMedicalInstitute,TheUniversityofChicago,929East57thStreet,Chicago,Illinois60637,USA.</auth-address><titles><title>Post-transcriptionalgeneregulationbymRNAmodifications</title><secondary-title>NatRevMolCellBiol</secondary-title><alt-title>Naturereviews.Molecularcellbiology</alt-title></titles><pages>31-42</pages><volume>18</volume><number>1</number><edition>2016/11/04</edition><keywords><keyword>5-Methylcytosine/metabolism</keyword><keyword>Adenosine/analogs&derivatives/metabolism</keyword><keyword>Animals</keyword><keyword>CellCycle/genetics</keyword><keyword>CellDifferentiation/genetics</keyword><keyword>CircadianRhythm/genetics</keyword><keyword>GeneExpressionRegulation</keyword><keyword>Humans</keyword><keyword>Methylation</keyword><keyword>NucleicAcidConformation</keyword><keyword>ProteinBiosynthesis</keyword><keyword>*RNAProcessing,Post-Transcriptional</keyword><keyword>RNAStability</keyword><keyword>RNA,Messenger/chemistry/genetics/*metabolism</keyword></keywords><dates><year>2017</year><pub-dates><date>Jan</date></pub-dates></dates><isbn>1471-0080<Electronic> 1471-0072<Linking></isbn><accession-num>27808276</accession-num><work-type>Review</work-type><urls><related-urls><url>/pubmed/27808276</url></related-urls></urls><custom2>5167638</custom2><electronic-resource-num>10.1038/nrm.2016.132</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Zhao,2017#8521"10]m6A生物学功能越来越多的证据表明m6A修饰在哺乳动物中发挥重要的生物功能。例如,在转录后水平上调控RNA的稳定性ADDINEN.CITEADDINEN.CITE.DATA[\o"Wang,2014#8489"11]、定位ADDINEN.CITEADDINEN.CITE.DATA[\o"Fustin,2013#8490"12]、运输、剪切ADDINEN.CITEADDINEN.CITE.DATA[\o"Molinie,2016#8494"13]和翻译ADDINEN.CITEADDINEN.CITE.DATA[\o"Meyer,2015#8491"14]。ClaudioR.等发现依赖METTL3的pri-miRNA甲基化,会促进DGCR8识别和加工,从而促进microRNA的成熟ADDINEN.CITE<EndNote><Cite><Author>Alarcon</Author><Year>2015</Year><RecNum>8493</RecNum><DisplayText>[15]</DisplayText><record><rec-number>8493</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8493</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Alarcon,C.R.</author><author>Lee,H.</author><author>Goodarzi,H.</author><author>Halberg,N.</author><author>Tavazoie,S.F.</author></authors></contributors><auth-address>LaboratoryofSystemsCancerBiology,RockefellerUniversity,1230YorkAvenue,NewYork,NewYork10065,USA.</auth-address><titles><title>N6-methyladenosinemarksprimarymicroRNAsforprocessing</title><secondary-title>Nature</secondary-title><alt-title>Nature</alt-title></titles><pages>482-5</pages><volume>519</volume><number>7544</number><edition>2015/03/25</edition><keywords><keyword>Adenosine/*analogs&derivatives/metabolism</keyword><keyword>BaseSequence</keyword><keyword>CellLine</keyword><keyword>GeneExpressionRegulation</keyword><keyword>Humans</keyword><keyword>Methylation</keyword><keyword>Methyltransferases/deficiency/metabolism</keyword><keyword>MicroRNAs/*chemistry/*metabolism</keyword><keyword>MolecularSequenceData</keyword><keyword>NucleicAcidConformation</keyword><keyword>*RNAProcessing,Post-Transcriptional</keyword><keyword>RNA-BindingProteins/metabolism</keyword><keyword>SubstrateSpecificity</keyword></keywords><dates><year>2015</year><pub-dates><date>Mar26</date></pub-dates></dates><isbn>1476-4687<Electronic> 0028-0836<Linking></isbn><accession-num>25799998</accession-num><work-type>ResearchSupport,U.S.Gov't,Non-P.H.S.</work-type><urls><related-urls><url>/pubmed/25799998</url></related-urls></urls><custom2>4475635</custom2><electronic-resource-num>10.1038/nature14281</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Alarcon,2015#8493"15]。此外,m6A识别蛋白HNRNPA2B1促进pri-miRNA加工成pre-miRNAADDINEN.CITEADDINEN.CITE.DATA[\o"Alarcon,2015#8495"16]。另外,环状RNA上m6A的修饰能促进环状RNA的翻译ADDINEN.CITEADDINEN.CITE.DATA[\o"Yang,2017#8505"17]。m6A修饰在基因表达调控中起着重要的作用,其调控机制的异常可能与人类疾病或癌症相关。目前发现m6A可能会影响精子发育〔ALKBH5,METTL3,Ythdc2〕、发育〔METTL3、FTO、ALKBH5〕、免疫〔METTL3〕、UV诱导的DNA损伤反应〔METTL3,FTO〕、肿瘤生成〔YTHDF2〕或转移〔METTL14〕、干细胞更新〔METTL14〕、脂肪分化〔FTO〕、生物节律、细胞发育分化、细胞分裂与其它的一些生命过程。例如,ALKBH5敲除的雄性小鼠增加了mRNA中的m<6>A修饰,其特点是凋亡影响减数分裂中期的精子细胞,引起生育能力受损ADDINEN.CITEADDINEN.CITE.DATA[\o"Zheng,2013#8501"7]。METTL3和METTL14增加弱精症精子的m6A水平ADDINEN.CITEADDINEN.CITE.DATA[\o"Yang,2016#8506"18],在生殖细胞中,METTL3的敲除严重抑制精子分化和减数分裂的发生,转录组和m6A分析显示精子发生相关基因的表达和选择性剪接发生了改变ADDINEN.CITEADDINEN.CITE.DATA[\o"Xu,2017#8509"19]。YTHDC2可促进靶基因的翻译效率,并降低其mRNA的丰度,在精子发生过程中起关键作用。当减数分裂开始时YTHDC2表达上调,YTHDC2敲除小鼠的生殖细胞没有经过偶线期的发育导致小鼠不育ADDINEN.CITE<EndNote><Cite><Author>Hsu</Author><Year>2017</Year><RecNum>8510</RecNum><DisplayText>[20]</DisplayText><record><rec-number>8510</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8510</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Hsu,P.J.</author><author>Zhu,Y.</author><author>Ma,H.</author><author>Guo,Y.</author><author>Shi,X.</author><author>Liu,Y.</author><author>Qi,M.</author><author>Lu,Z.</author><author>Shi,H.</author><author>Wang,J.</author><author>Cheng,Y.</author><author>Luo,G.</author><author>Dai,Q.</author><author>Liu,M.</author><author>Guo,X.</author><author>Sha,J.</author><author>Shen,B.</author><author>He,C.</author></authors></contributors><auth-address>DepartmentofChemistryandInstituteforBiophysicalDynamics,TheUniversityofChicago,Chicago,IL60637,USA. HowardHughesMedicalInstitute,TheUniversityofChicago,Chicago,IL60637,USA. CommitteeonImmunology,TheUniversityofChicago,Chicago,IL60637,USA. StateKeyLaboratoryofReproductiveMedicine,DepartmentofHistologyandEmbryology,NanjingMedicalUniversity,Nanjing211166,China. DepartmentofBiochemistryandMolecularBiology,TheUniversityofChicago,Chicago,IL60637,USA.</auth-address><titles><title>Ythdc2isanN6-methyladenosinebindingproteinthatregulatesmammalianspermatogenesis</title><secondary-title>CellRes</secondary-title><alt-title>Cellresearch</alt-title></titles><edition>2017/08/16</edition><dates><year>2017</year><pub-dates><date>Aug15</date></pub-dates></dates><isbn>1748-7838<Electronic> 1001-0602<Linking></isbn><accession-num>28809393</accession-num><urls><related-urls><url>/pubmed/28809393</url></related-urls></urls><electronic-resource-num>10.1038/cr.2017.99</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Hsu,2017#8510"20]。在DNA损伤反应中,METTL3可促进DNA聚合酶κ〔Polκ〕与核酸剪切修复途径快速定位到UV引起的DNA损伤位点,当缺失METTL3时,细胞无法迅速修复UV照射引起的突变,并且对UV照射更加敏感[25]。在淋巴细胞性小鼠过继转移模型中,Mettl3缺陷通过影响mRNAm6A修饰,降低SOCS家族mRNA衰减,增加mRNA和蛋白表达水平,从而抑制IL-7介导的STAT5活性和T细胞内稳态增殖和分化,进而抑制肠炎的发生ADDINEN.CITEADDINEN.CITE.DATA[\o"Li,2017#8518"21]。在肝癌中,METTL14通过调控pri-miRNA的m6A修饰,影响MiR-126的生成加工,从而抑制肝癌的转移ADDINEN.CITEADDINEN.CITE.DATA[\o"Ma,2017#8511"22]。在乳腺癌细胞中,低氧刺激能促进依赖低氧诱导因子HIF的ALKBH5的表达,而ALKBH5过表达降低了NANOGmRNA的m6A修饰,从而稳定mRNA提高NANOG的表达水平,最终增加乳腺癌干细胞所占的比例ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhang,2016#8512"23]。此外,低氧诱导乳腺癌细胞中依赖ZNF217的NANOG和KLF4的mRNAm6A甲基化抑制,且ALKBH5敲除显著降低免疫缺陷小鼠乳腺癌的肺转移ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhang,2016#8513"24]。在肺癌中,METTL3能够促进肺腺癌细胞的生长、生存和侵袭,但还不清楚它是否作为m6A调节器或效应器发挥作用ADDINEN.CITEADDINEN.CITE.DATA[\o"Lin,2016#8514"25]。在急性髓细胞白血病〔AML〕患者中,m<6>A调控基因的突变或拷贝数变化与TP53突变存在密切联系,且m<6>A调控基因的改变与AML不良预后相关ADDINEN.CITE<EndNote><Cite><Author>Kwok</Author><Year>2017</Year><RecNum>8515</RecNum><DisplayText>[26]</DisplayText><record><rec-number>8515</rec-number><foreign-keys><keyapp="EN"db-id="f2s2fdzw5a5wp6epsfu5dtfpwwfa0p2vw9ev">8515</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Kwok,C.T.</author><author>Marshall,A.D.</author><author>Rasko,J.E.</author><author>Wong,J.J.</author></authors></contributors><auth-address>Gene&StemCellTherapyProgram,CentenaryInstitute,UniversityofSydney,Camperdown,2050,Australia. GeneRegulationinCancerLaboratory,CentenaryInstitute,UniversityofSydney,Camperdown,2050,Australia. SydneyMedicalSchool,UniversityofSydney,Camperdown,NSW,2006,Australia. CellandMolecularTherapies,RoyalPrinceAlfredHospital,Camperdown,2050,Australia. Gene&StemCellTherapyProgram,CentenaryInstitute,UniversityofSydney,Camperdown,2050,Australia..au. GeneRegulationinCancerLaboratory,CentenaryInstitute,UniversityofSydney,Camperdown,2050,Australia..au. SydneyMedicalSchool,UniversityofSydney,Camperdown,NSW,2006,Australia..au.</auth-address><titles><title>Geneticalterationsofm6Aregulatorspredictpoorersurvivalinacutemyeloidleukemia</title><secondary-title>JHematolOncol</secondary-title><alt-title>Journalofhematology&oncology</alt-title></titles><pages>39</pages><volume>10</volume><number>1</number><edition>2017/02/06</edition><dates><year>2017</year><pub-dates><date>Feb02</date></pub-dates></dates><isbn>1756-8722<Electronic> 1756-8722<Linking></isbn><accession-num>28153030</accession-num><work-type>Letter</work-type><urls><related-urls><url>/pubmed/28153030</url></related-urls></urls><custom2>5290707</custom2><electronic-resource-num>10.1186/s13045-017-0410-6</electronic-resource-num><language>eng</language></record></Cite></EndNote>[\o"Kwok,2017#8515"26]。此外,FTO在AML中高表达,它通过降低mRNA转录本中的m<6>水平,调节ASB2和RARA等靶点的表达,增强了白血病癌基因介导的细胞转化和白血病形成,并抑制全反式维甲酸<ATRA>诱导的AML细胞分化ADDINEN.CITEADDINEN.CITE.DATA[\o"Li,2017#8516"27]。在脂肪形成过程中,FTO表达与m6A水平成负相关,促进脂肪形成ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhao,2014#8497"3]。在胶质细胞瘤样细胞中,ALKBH5通过lncRNAFOXM1介导FOXM1基因pre-mRNA上的m6A修饰维持胶质瘤细胞的成瘤性ADDINEN.CITEADDINEN.CITE.DATA[\o"Zhang,2017#8507"28]。此外,甲基转移酶METTL3或METTL14的敲除,能够改变m6A的富集和ADAM19的表达,极大地促进了胶质瘤细胞的生长、自我更新和肿瘤形成ADDINEN.CITEADDINEN.CITE.DATA[\o"Cui,2017#8508"29]。图2m6ARNA修饰和介导的功能ADDINEN.CITEADDINEN.CITE.DATA[\o"Cao,2016#8520"30]m6A的研究方向主要是通过研究m6A修饰相关的甲基化、去甲基化酶和识别蛋白的功能,进而研究m6A修饰的生物学功能和作用机制：一般通过敲除m6A酶分子,研究下游功能基因分子的表达和m6A甲基化情况,通过介导相关基因异常〔可变剪切、稳定性、翻译、miRNA调控〕影响细胞表型和功能特征。m6A修饰图谱构建与作用机制：通过m6A甲基化测序〔MeRIP-Seq,miCLIP〕构建疾病细胞模型或者发病组织的m6A修饰谱,分析m6A的motif,peaks数量与分布,Peak关联基因的特征,联合RNA-seq研究m6A甲基化与表达的关系。m6A研究思路m6A研究方案疾病样本疾病样本VS正常样本RNA-seqMeRIP-seqm6A修饰图谱分析m6A修饰差异基因分析差异表达基因m6A修饰特征分析差异表达基因关联分析MeRIP-PCR、qPCR验证方案一TCGA等数据库筛选异常表达的m6A相关基因〔疾病vs正常〕临床样本qPCR验证目标TCGA等数据库筛选异常表达的m6A相关基因〔疾病vs正常〕临床样本qPCR验证目标基因肿瘤细胞中干扰目标基因MTT、流式、transwell等检测细胞增殖、凋亡、侵袭和迁移MeRIP-SeqRNA-seq筛选下游基因IP/pulldown验证目标基因通过m6A调控下游基因研究案例研究案例一〔m6A修饰图谱分析〕BerulavaT,RahmannS,RademacherK,Klein-HitpassL,HorsthemkeB:N6-adenosinemethylationinMiRNAs.PLoSOne2015,10<2>:e0118438.在许多不同种类的RNA中,都已观察到N6-腺苷<m6A>的甲基化,但其在microRNAs中还没有被研究。研究者在FTO1C1,FTO2D4和FTO3C3细胞系中,通过敲除m6A甲基转移酶FTO筛选到表达差异的microRNA,说明miRNA受m6A甲基化的调控。进一步通过MeRIP-Seq发现相当一部分的microRNA具有m6A修饰。通过motif分析,他们发现了区分甲基化和非甲基化microRNA的一致序列。该文章所述的表观遗传修饰在基因表达的转录后调控的复杂性上增加了一个新的层次。图1FTO敲除对甲基化的miRNAs的稳定状态的影响。研究案例二〔机制研究〕IF=13.2MaJZ,YangF,ZhouCC,LiuF,YuanJH,WangF,WangTT,XuQG,ZhouWP,SunSH:METTL14suppressesthemetastaticpotentialofhepatocellularcarcinomabymodulatingN6-methyladenosine-dependentprimaryMicroRNAprocessing.Hepatology2017,65<2>:529-543.m6A修饰已被证明具有重要的生物学功能,但其在癌症上的作用还未得到较好的研究。为探索m6A修饰是否参与肝癌的调控,作者利用试剂盒检测发现m6A整体甲基化在肝癌下调,分离RNA做m6A免疫印迹验证m6A水平在肝癌中下调。为研究哪些因子导致m6A在肝癌的下调,作者在20例肝癌与癌旁中检测m6A甲基化酶和去甲基酶的表达,发现METTL14在肝癌显著下调,进一步通过130例病人分析发现METTL14作为肝癌预后因子,细胞实验发现其敲除可增强肝癌转移。接下来作者研究METTL14抑制肝癌转移的机制,由于已有研究显示m6A修饰能够增强DGCR8蛋白识别pri-miRNAs,促进miRNAs的成熟,因此作者在METTL14敲除细胞中检测miRNA和pri-miRNAs的表达,发现miR-126下调。通过免疫沉淀反应发现METTL14与DGCR8依赖RNA相互作用,且CLIP实验显示DGCR8与m6A-RNA相互作用且敲除METTL14后结合作用降低,说明METTL14通过m6A修饰促进DGCR8识别pri-miR-126.最后细胞实验证明MiR-126能够回复METTL14的肝癌细胞转移抑制功能,证明了METTL14通过m6A修饰促进miR-126加工,从而抑制肝癌细胞转移。图1METTL14在肝癌中下调图2METTL14依赖的m6A通过DGCR8调控miR-126加工研究案例三〔机制研究〕IF=27.4ZhangS,ZhaoBS,ZhouA,LinK,ZhengS,LuZ,ChenY,SulmanEP,XieK,BoglerOetal:m6ADemethylaseALKBH5MaintainsTumorigenicityofGlioblastomaStem-likeCellsbySustainingFOXM1ExpressionandCellProliferationProgram.CancerCell2017,31<4>:591-606e596.DNA甲基化异常是胶质瘤的表观遗传调控因子,但RNA甲基化在肿瘤包括恶性胶质瘤〔GBM〕中的调控还尚未清楚。为研究m6A调节可能导致GBM患者临床疗效不佳,作者通过TCGA数据库,发现ALKBH5在恶性胶质瘤样干细胞〔GSCs〕中高表达且与GBM病人不良预后相关,干扰ALKBH5降低GSCs细胞的自我更新能力且抑制GSCs增殖,进一步体内验证敲除ALKBH5可抑制肿瘤生长。为研究ALKBH5的m6A作用机制,作者利用芯片和m6A-seq筛选到胶质瘤增殖相关的FOXM1,最后通过qPCR、WB、免疫荧光、核质分离WB/qPCR、RIP和MeRIP等实验证明ALKBH5通过去甲基化初期转录本调节FOXM1在GSCs中的表达。为研究ALKBH5对FOXM1的作用是否受其他因子的调节,作者研究了FOXM1的邻近基因,发现lncRNAFOXM1-AS与FOXM1序列互补,且共表达、共定位,进一步通过RIP,RNApulldown等实验证明lncRNAFOXM1-AS促进ALKBH5和FOXM1初级转录本的相互作用。最后通过细胞实验进一步验证ALKBH5在lncRNAFOXM1-AS的作用下维持FOXM1的表达和细胞增殖程序,从而维持GSCs的干性。图1ALKBH5敲除细胞中m6A修饰的特征和基因表达的变化ADDINEN.REFLIST1. 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