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Journal of Southern Medical University logoLink to Journal of Southern Medical University
. 2022 May 20;42(5):690–697. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2022.05.09

t(8;21)急性髓系白血病中RNA N6-甲基腺嘌呤（m6A）的修饰特征

Characteristics of N6-methyladenosine modification patterns in t(8;21) acute myeloid leukemia

Yanan WEN 1,2, Shu FANG 2,3, Jingjing YANG 1,2, Hao WANG 1,2, Yifan JIAO 1,2, Nan WANG 1,2, Yan WEI 1,2, Lili WANG 2, Liping DOU 2,*
PMCID: PMC9178644  PMID: 35673912

Abstract

Objective

To investigate the relationship between AML1-ETO (AE) fusion gene and intracellular N6-methyladenosine (m6A) modification pattern in t(8;21) acute myeloid leukemia (AML).

Methods

RNA m6A sequencing was performed in SKNO-1 and AE knockdown SKNO-1 (SKNO-1 siAE) cells using RNA-protein co-immunoprecipitation and high-throughput sequencing (methylated RNA immunoprecipitation sequencing, MeRIP-Seq) to analyze the changes in m6A modification of the entire transcriptome. Transcriptome sequencing (RNA-seq) was performed using high-throughput sequencing. The differentially modified mRNAs were further functionally annotated by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The changes in m6A-related enzyme expressions were detected using real-time PCR.

Results

A total of 26 441 genes were identified in AE knockdown AML cells and AE-expressing cells, containing 72 036 m6A peaks. AE knockdown caused a reduction of the number of intracellular m6A peaks from 37 042 to 34 994, among which 1278 m6A peaks were significantly elevated and 1225 were significantly decreased; 1316 genes with newly emerged m6A modification were detected and 1830 genes lost m6A modification after AE knockdown. The differential peaks were mainly enriched in pathways involving cancer and human T-lymphocytic leukemia virus I. RNA-seq results showed that 2483 genes were up-regulated and 3913 genes were down-regulated after AE knockdown. The combined analysis of MeRIP-Seq and RNA-Seq results revealed relatively high expression levels of m6A-modified genes as compared with the genes without m6A modification (SKNO-1: 0.6116±1.263 vs 2.010±1.655, P < 0.0001; SKNO-1 siAE: 0.5528±1.257 vs 2.067±1.686, P < 0.0001). The m6A modified genes located in the 3'UTR or 5 'UTR had significantly higher expression levels than those located in exonic regions (SKNO-1: 2.177± 1.633 vs 1.333 ± 1.470 vs 2.449 ± 1.651, P < 0.0001; SKNO-1 siAE: 2.304 ± 1.671 vs 1.336 ± 1.522 vs 2.394 ± 1.649, P < 0.05). Analysis of RNA-seq data identified 3 m6A-related enzymes that showed significantly elevated mRNA expression after AE knockdown, namely WTAP, METTL14, and ALKBH5 (P < 0.05), but the results of real-time PCR showed that the expressions of WTAP and ALKBH5 were significantly increased while the expression of METTL14 was lowered after AE knockdown (P < 0.05).

Conclusion

AE knockdown results in differential expressions of m6A-associated enzymes, suggesting that the AE fusion gene regulates the expression of one or more m6A-associated enzymes to control cellular methylation levels.

Keywords: AML1-ETO, acute myeloid leukemia, N6-methyladenosine

急性髓系白血病(AML)是一种以髓系干/祖细胞克隆性增殖和分化受阻为特征的血液系统恶性疾病[1]。AML1-ETO（AE）来源于（t 8；21）（q22；q22）染色体易位，见于约4%~8%的AML[2, 3]。除了通过其RUNT结构域直接与靶基因结合外[4]，AML1-ETO还可以被招募而形成稳定的AETFC复合物与DNA结合[3]，使其在转录水平上通过与靶基因结合而调控转录。AE的缺失会导致转录因子结合的广泛改变，进而改变基因表达，并损害AML的生长[5, 6]。尽管可以确定因AE敲除改变的基因，但鉴于基因表达的调控可以发生在转录和转录后水平上，因此无法确定这些改变是直接还是间接发生的。而AE是否以及如何在转录后水平调控基因表达尚不清楚。

N6-甲基腺嘌呤（m6A）是高等生物mRNA和IncRNA上甲基化修饰最普遍的存在方式[7, 8]。m6A通过调节RNA稳定性和蛋白质翻译速率而在转录后水平上调节基因表达并显著影响癌症的发展[9]。m6A甲基化过程表现出动态和可逆的特征[10]，它可以由甲基转移酶复合物（如WTAP、METTL3、METTL14）安装[11, 12]，同时也可以被去甲基化酶（如FTO、ALKBH5）清除[13, 14]，并被m6A结合蛋白（如YTHDF1/2/3、YTHDC1/2和IGF2BP1/2/3）识别[15]。研究发现甲基转移酶METTL14在伴有（t 8;21）、（t 11q23）或（t 15;17）的AML中高表达，通过改变m6A修饰模式促进白血病的发展[15, 16]。

因此本研究将AE依赖的转录调控与m6A相结合，通过对AE敲除前后的AML细胞进行m6A测序，分析细胞内的m6A修饰特征，观察AE在转录后水平上通过改变m6A修饰模式对基因表达的影响。

1. 材料和方法

1.1. 试剂与仪器

RPMI 1640培养液、胎牛血清、青霉素链霉素双抗混合液（Gibco），mRNA提取试剂盒Magnetic mRNA Isolation Kit S1550S[NEB（北京）有限公司]，HybondN+膜（GE Healthcare），Dot Blot（170-3938）（Bio-Rad），m6A抗体（Proteintech），二抗抗兔抗体（CST），ChemiDocTM成像系统（BIO-RAD）。

1.2. 细胞培养

SKNO-1与SKNO-1 SIAE细胞株由301医院血液科实验室提供，自液氮复苏后将细胞培养于含10%胎牛血清、1%双抗的RPMI 1640培养液的培养皿中，放置培养箱培养（37 ℃、5% CO2、饱和湿度），每2~3 d换液传代1次。

1.3. MeRIP-seq和RNA-seq

利用MeRIP-Seq在AE(+)和敲除AE的AML细胞系中进行RNAm6A测序，分析整个转录组m6A修饰的变化。利用高通量测序技术进行转录组测序（RNA-seq）。基因测序由杭州联川生物技术股份有限公司进行。使用Trizol试剂提取总RNA后，使用Oligo-dT磁珠对total RNA带有polyA的mRNA进行富集。对磁珠进行富集，得到带有polyA的mRNA。之后加入片段化试剂，将完整的mRNA进行片段化，片段化长度约为100 nt。将片段化后的RNA分成2份。一份加入带有预混好的m6A抗体免疫磁珠，对含有m6A甲基化的mRNA片段进行富集。另一份作为对照，直接构建常规的转录组测序文库。对m6A抗体免疫磁珠进行富集，带有m6A的mRNA片段进行回收后，按照转录组的建库流程构建常规的测序文库。分别将构建好的2个测序文库，即m6A-seq library（IP）和RNA-seq library（input）分别进行高通量测序，测序平台为Illumina NovaseqTM 6000，测序模式为150 PE。

1.4. 实时荧光定量PCR

SKNO-1与SKNO-1 siAE培养1周后，通过trizol法提取总RNA。紫外分光光度计测定RNA浓度。利用快速逆转录试剂盒制备cDNA。KAPA SYBR® FAST qPCR试剂盒进行实时定量荧光PCR。以GAPDH作为对照。20 μL反应体系，95 ℃ 20 s；95 ℃ 3 s，60 ℃ 20 s，72 ℃ 20 s，40个循环。所用引物如表 1。

1.

引物序列

Primer sequences

Gene Forward primers sequences Reverse primers sequences
WTAP GCTTCTGCCTGGAGAGGATT TGCAGACTCCTGCTGTTGTT
METTL14 AGAGAACAAAGGAACACTGCCT AATGAAGTCCCCGTCTGTGC
ALKBH5 CGGCGAAGGCTACACTTACG CCACCAGCTTTTGGATCACCA
GAPDH GGAGCGAGATCCCTCCAAAAT GGCTGTTGTCATACTTCTCATGG
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1.5. 数据分析

1.5.1. MeRIP-seq&RNA-seq数据分析

Cutadapt以及本地perl脚本去除低质量序列、污染序列以及测序仪接头序列，得到CleanData。使用fastp软件对CleanData进行质控。使用HISAT2的默认参数将reads比对到参考基因组上。exomePeak和ChIPseeker进行Peak calling分析和Peak注释。MEME和HOMER对富集区域进行motif分析。基因定量软件为StringTie，归一化方式为FPKM。基因差异分析采用R包edgeR。ggplot2包对GO富集分析结果以散点图展示，Rich factor表示位于该GO的差异基因个数/位于该GO的总基因数。

1.5.2. 统计学分析

采用IBM SPSS Statistics 24.0及GraphPad Prism 9软件进行统计分析。定量资料采用均数±标准差表示，独立样本t检验用于符合正态分布定量资料的组间比较；Mann-Whitney用于不符合正态分布定量资料的组间比较，P < 0.05为差异有统计学意义。

2. 结果

2.1. SKNO-1与SKNO-1 siAE细胞系的m6A修饰

通过MeRIP- Seq技术对AE(+)的AML细胞系SKNO-1与敲除AE的SKN0-1 siAE细胞系进行RNA m6A甲基化测序，测序结果的主成分分析显示，SKN0-1 siAE细胞系的2个样本可以与SKNO-1细胞系的2个样本区别开，具有可比性（图 1A）。为了分析m6A在两个细胞系转录组中的分布，根据其在mRNA上的位置，将m6A peak划分为5′非翻译区（5'UTR）、蛋白质编码区（CDS）及3′非翻译区（3'UTR），m6A peak主要在2个细胞系的外显子区及3'UTR富集（图 1B、C）。峰密度分析也显示m6A peak主要富集于CDS区与3'UTR（图 1D）。Motif分析显示，在2个细胞系内均有m6A结合基序RRACH（图 1E）。在测序结果中，共检测到26 441个基因，包含了72 036个m6A peak。其中有2164个基因仅在SKNO-1细胞系中表达，3395个基因仅在SKNO-1 siAE细胞系中表达，12 451个基因在两细胞系共表达（图 1F）。

1.

1

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SKNO-1与SKNO-1 siAE细胞中的m6A修饰模式

Overview of m6A modification patterns in SKNO-1 and SKNO-1 siAE cells. A: Principle component analysis reveals that the biological duplicated samples of SKNO-1 siAE and SKNO-1 cells are comparable. B: m6A peak density along the transcript. The transcipts were divided into three parts, including 5' untranslated region (5'UTR), coding sequence (CDS), and 3'UTR. C, D: m6A peaks are mostly enriched in 3'UTR and CDS of the transcripts. E: The top two motifs identified from m6A peaks enriched in SKNO-1 siAE and SKNO-1 cells. F: Distribution of m6A-modified genes in SKNO-1 and SKNO-1 siAE cells.

2.2. m6A动态分析

AE敲除后细胞内的m6A peak数量由37 042个变成34 994个（图 2A）。其中1278个peak显著上调，1225个peak显著下调，20 827个peak无显著变化（P < 0.05，log2fc>1，图 2B）。分析显示AE敲除后增多的peak主要集中在CDS区与3'UTR区，降低的peak则主要集中在CDS区（图 2C）。AE敲除后新出现了1316个m6A修饰的基因，1830个基因失去了m6A修饰（图 2D）。KEGG通路分析的结果显示，差异的peak主要在癌症、人类T淋巴细胞白血病病毒Ⅰ、嘌呤代谢、血小板活化等通路中富集（图 2E）。

2.

2

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m6A修饰的mRNA差异m6A peak分析

Analysis of differentially methylated m6A modified mRNAs. A: Fold enrichment of all m6A peaks in SKNO-1 and SKNO-1 siAE. B: Volcano plot of differential m6A peaks. Red and blue dots represent hyper- and hypo-methylated peaks, respectively. C: Distribution of the differential peaks in the transcripts of SKNO-1 and SKNO-1 siAE cells. D: Overlapped m6A labeled genes in SKNO-1 and SKNO-1 siAE cells. E: Pathway analysis of differential peaks in SKNO-1 siAE as compared with SKNO-1 cells.

2.3. AE敲除后基因表达水平变化

对两个细胞系的基因表达水平进行比较，结果显示，敲除AE融合基因后出现6000多个差异表达的基因（图 3A，log2 fc>1.5，P < 0.05），其中2483个基因表达上调，3913个基因表达下降（图 3B）。差异表达的基因分布于癌症中的转录失调、肿瘤坏死因子、调节干细胞多能性、癌症通路等多条通路中（图 3C）。GO分析表明，这些差异表达的基因参与了包括质膜形成、多细胞生物发育等多种生理过程（图 3D）。

3.

3

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SKNO-1与SKNO-1 siAE细胞的差异基因分析

Analysis of differentially expressed genes by RNA-seq. A: Volcano plot of differently expressed genes. B: Distribution of differentially expressed genes. C: Pathway analysis of the differentially expressed genes in SKNO-1 and SKNO-1 siAE cells. D: GO enrichment of the differentially expressed genes in SKNO-1 and SKNO-1 siAE cells.

2.4. RNA-seq与m6A-seq的联合分析

MeRIP-Seq和RNA-Seq联合分析结果显示，在AE敲除和未敲除中，与非m6A修饰的基因相比，m6A所修饰的基因表达水平更高（SKNO-1:0.6116 ± 1.263 vs 2.010 ± 1.655，P < 0.0001；SKNO-1 siAE: 0.5528 ± 1.257 vs 2.067±1.686，P < 0.0001，图 4A）。GO分析显示，转录组参与转录调控、信号诱导等生物过程，其基因产物在细胞质、胞浆与细胞核等部位发挥作用，影响着蛋白质、离子、DNA结合等功能（图 4B）。当m6A修饰落在不同区域时，对于仅有一个m6A修饰位点的基因，若其在外显子区被m6A修饰，则表达量低于3'UTR或5'UTR m6A修饰的基因（SKNO-1：1.973±1.737 vs 1.018±1.380 vs 2.267±1.872，P < 0.0001；SKNO-1 siAE：2.181±1.826 vs 0.9823±1.424，P < 0.0001，0.9823±1.424 vs 2.065±1.860，P < 0.0001，2.181±1.826 vs 2.065±1.860，NS，图 4C）。统计所有被m6A修饰的基因，也表现出相同的趋势（SKNO-1:2.177±1.633 vs 1.333±1.470 vs 2.449±1.651，P < 0.0001；SKNO-1 siAE: 2.304±1.671 vs 1.336±1.522 vs 2.394±1.649，P < 0.05，图 4D）。对于m6A修饰的数量与基因表达量关系的分析显示，与仅有一个m6A修饰位点的基因相比，拥有更多m6A修饰位点的基因其表达量也相对更高（图 4E）。

4.

4

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mRNAm6A甲基化测序与RNA测序联合分析

Conjoint analysis of MeRIP-seq and RNA-seq data. A: Expression levels of genes with and without m6A modification in SKNO-1 and SKNO-1 siAE cells (****P < 0.0001). B: GO analysis of conjoint analysis. C: Analysis of expression levels of genes with one m6A peak in different locations of mRNA (**P < 0.01, ****P < 0.0001). D: Analysis of expression levels of all genes with m6A modification in different locations of mRNA(****P < 0.0001). E: Analysis of expression levels of genes with different numbers of m6A peaks (***P < 0.001, ****P < 0.0001). F: Analysis of expression level of m6A writers and erasers by RNA-seq (*P < 0.05, **P < 0.01, ***P < 0.001). G: Analysis of expression level of m6Awriters and erasers by RT-qPCR in SKNO-1 and SKNO-1 siAE cells (*P < 0.05, **P < 0.01, ***P < 0.001).

2.5. AE敲除后m6A相关酶水平的变化

AE敲除后，测序结果显示，AE敲除后2种甲基转移酶WTAP、METTL14与去甲基化酶ALKBH5表达量均升高（WTAP: 5.36 ± 0.5657 vs 13.19 ± 0.3253，METTL14：2.850 ± 0.1556 vs 8.815 ± 1.761，ALKBH5: 13.70±0.4596 vs 39.84±6.067，P < 0.05，图 4F）。qPCR检测敲除AE前后METTL14、WTAP、ALKBH5的表达水平，结果显示WTAP表达量升高1.7倍、ALKBH5表达量升高2.4倍，而METTL14表达量降低至0.93倍（图 4G）。

3. 讨论

M6A修饰作为真核生物中最普遍的转录后修饰不改变碱基配对和编码, 但通过不同的甲基转移酶、去甲基化酶、阅读蛋白和相关复合物相互作用，在多个水平上广泛影响基因表达[17]。因此，动态m6A修饰对许多正常生物过程以及癌症的发生、进展、转移、耐药性和癌症复发至关重要。以往对于AML中AML1-ETO融合基因与m6A修饰的研究大多集中于这二者独立的作用机制。如AE调节的转录对白血病发生发展的影响[18-20]，或m6A及m6A相关酶在白血病中的作用[21-24]。鲜少有对AE与m6A之间关系的研究。因此本研究希望观察AE在转录后水平上通过改变m6A修饰模式对基因表达的影响，探索AE依赖的转录与m6A修饰之间的联系。

本研究发现m6A peak主要在敲除AE和表达AE的外显子区及3'UTR富集[25, 26]，这与此前真核生物细胞内有关m6A的研究结果一致。且Motif分析显示在两个细胞系内均有m6A结合基序RRACH，符合先前研究中对m6A共识结合基序[G/A/U][G>A]m6AC[U>A>C] 的认识[27-30]。这证明本研究结果中对m6A的研究是准确可靠的。

既往研究证实，在SKNO-1细胞中抑制AML-ETO可导致细胞分化和白血病细胞生长抑制[31]，通过siRNA将AML1-ETO敲除可使SKNO-1细胞系对诱导细胞分化的TGFβ1敏感[32]，克隆形成减少，抑制细胞增殖并诱导衰老[33]。本研究在敲除AE和表达AE的AML细胞系中进行RNA m6A甲基化测序，结果显示AE敲除后新出现了1316个m6A修饰的基因，1830个基因失去了m6A修饰。增多的peak主要集中在CDS区与3'UTR区，降低的peak则主要集中在CDS区。AE的敲除对细胞内的m6A修饰数量和位点均造成了影响，显示出AE对m6A修饰模式的调控作用。结合敲除AML1-ETO后细胞内m6A修饰模式的改变，可以推测AML1-ETO这种对细胞功能的影响或许与其改变m6A修饰模式的能力有关。

RNA-seq结果的GO分析显示差异表达的基因与m6A peak富集在干细胞多能性的调节、癌症中的转录失调等多条通路中，提示AE与m6A对癌症发生发展的重要性。KEGG通路分析显示差异的m6A peak与基因在转化生长因子、肿瘤坏死因子等信号通路中富集，其与细胞生长、凋亡、分化与肿瘤发生发展有着密切联系[34, 35]。MeRIP-Seq和RNA-Seq联合分析提示，是否有m6A修饰、m6A修饰位点的数量与位置对调控基因表达水平起着重要的作用。当t（8；21）AML发生时，AML1-ETO或许是通过在这些通路上调控基因的m6A修饰状态进而调节其表达，促进AML的发展。

此外，在RNA-seq与实时荧光定量PCR的结果中均检测到AE敲除后甲基化酶WTAP、METTL14和去甲基化酶ALKBH5的表达变化，这提示AE或许通过调控一种或多种m6A相关酶的表达控制细胞内的甲基化水平。其中甲基转移酶WTAP不止作为甲基转移酶复合物的重要组成部分[11, 36]，其自身也作为一种致癌因子在体内起着重要的作用[37]，并且与正常细胞相比，WTAP在急性髓系白血病细胞中的表达高于正常水平[38]。AE的敲除所造成的m6A相关酶差异表达展示了AE对于细胞内m6A相关酶的影响，推测AML1-ETO融合基因或许可以调控一种或多种m6A相关酶的表达控制细胞内的甲基化水平，影响m6A修饰模式，从而起到调节白血病细胞侵袭性生长的作用。这或许能为m6A调节剂治疗AE（+）的AML提供新的思路。

Biography

温亚男，在读硕士研究生，E-mail: wyn_15163875680@163.com

Funding Statement

国家自然科学基金（82070178，81770203，81700122，81270610）；解放军总医院转化项目（ZH19003）；解放军总医院医疗大数据与人工智能研发项目（2019MBD-016）

Supported by National Natural Science Foundation of China (82070178, 81770203, 81700122, 81270610)

Contributor Information

温 亚男 (Yanan WEN), Email: wyn_15163875680@163.com.

窦 立萍 (Liping DOU), Email: lipingruirui@163.com.

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