南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (5): 690-697.doi: 10.12122/j.issn.1673-4254.2022.05.09

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t(8;21)急性髓系白血病中RNA N6-甲基腺嘌呤（m6A）的修饰特征

温亚男，方 姝，杨晶晶，王 昊，焦一帆，王 楠，魏 岩，王莉莉，窦立萍   

  1. 解放军医学院，北京 100853；中国人民解放军总医院第五医学中心血液病医学部，北京 100853；南开大学，天津 300071
  • 出版日期:2022-05-20 发布日期:2022-06-02

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

WEN Yanan, FANG Shu, YANG Jingjing, WANG Hao, JIAO Yifan, WANG Nan, WEI Yan, WANG Lili, DOU Liping   

  1. Medical School of Chinese PLA, Beijing 100853, China; Department of Hematology, Fifth Medical Center of Chinese PLA General Hospital, Beijing 100853, China; Nankai University, Tianjin 300071, China
  • Online:2022-05-20 Published:2022-06-02

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摘要： 目的 研究（t8；21）急性髓系白血病（AML）中AML1-ETO（AE）融合基因与细胞内N6-甲基腺嘌呤（m6A）修饰的关系。方 法 利用RNA-蛋白免疫共沉淀和高通量测序技术（MeRIP-Seq）在AE（+）和敲除AE的AML细胞系中进行RNA m6A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）。进一步通过GO分析、KEGG通路富集分析对差异修饰的 mRNA 进行功能注释。实时荧光定量PCR检测m6A相关酶表达量变化。结果 RNA m6A甲基化测序在敲除AE和表达AE的AML细胞系中共检测到26 441个基因，包含了72 036个m6A peak。AE敲除后细胞内m6A peak的数目由37 042个变成34 994个，其中有1278个m6A peak升高，1225个下降。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。差异的peak主要在癌症、人类T淋巴细胞白血病病毒Ⅰ等通路中富集。RNA-seq结果显示，AE敲除后有2483个基因表达上调，3913个基因表达下调。MeRIP-Seq和RNA-Seq联合分析结果显示，与非m6A修饰的基因相比，m6A所修饰的基因表达水平均相对较高（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）。m6A修饰位于3'UTR或5'UTR的基因较位于外显子区的基因表达量更高（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）。RNA-seq结果显示，有3 种m6A相关酶METTL14、WTAP、ALKBH5的mRNA表达升高（WTAP:5.36±0.5657 vs 13.19±0.3253，METTL14:2.850±0.1556vs 8.815±1.761，ALKBH5:13.70±0.4596 vs 39.84±6.067，P<0.05）。实时荧光定量PCR检测METTL14、WTAP、ALKBH5的表达量变化发现敲除AE后WTAP、ALKBH5的表达量升高，而METTL14的表达量降低（P<0.05）。结论 AE敲除造成m6A相关酶差异，推测AE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。

关键词: AML1-ETO；急性髓系白血病；N6-甲基腺嘌呤（m6A）

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.

Key words: AML1-ETO; acute myeloid leukemia; N6-methyladenosine