METTL3介导ITGB3的m6A甲基化修饰促进巨核细胞分化*

doi:10.3969/j.issn.1671-2587.2025.05.003

临床输血与检验 ›› 2025, Vol. 27 ›› Issue (5): 594-603.DOI: 10.3969/j.issn.1671-2587.2025.05.003

METTL3介导ITGB3的m⁶A甲基化修饰促进巨核细胞分化^*

侯晓宇^1,2, 夏文军¹, 陈睿驰¹, 孙登莲¹, 吴煌¹, 文爱清^1,2

¹陆军军医大学大坪医院输血医学科,创伤与化学中毒全国重点实验室,重庆 400042;
²重庆大学生物与工程学院,重庆 400044

收稿日期:2025-09-02 修回日期:2025-09-12 出版日期:2025-10-20 发布日期:2025-10-11
通讯作者: 文爱清,主要从事危重症输血、止凝血障碍、血小板生成调控方面研究,（E-mail）wenaiqing@tmmu.edu.cn。共同通信作者：吴煌,主要从事RNA调控与血小板生成方面研究,（E-mail）huangwu@tmmu.edu.cn。
作者简介:侯晓宇,主要从事生物与医药方面研究,（E-mail）xiaoyu_hou2001@163.com。
基金资助:
^*本课题受国家自然科学基金面上项目（No.82472205、No.82170134、No.82172137）、陆军特色医学中心基础研究项目（No.ZXJCYJ202507）资助

METTL3-mediated m⁶A Methylation Modification of ITGB3 Promotes Megakaryocyte Differentiation

HOU Xiaoyu^1,2, XIA Wenjun¹, CHEN Ruichi¹, SUN Denglian¹, WU Huang¹, WEN Aiqing^1,2

¹Department of Transfusion Medicine, State Key Laboratory of Trauma and Chemical Poisoning, Daping Hospital, Army Medical University, Chongqing 400042;
²Bioengineering College of Chongqing University, Chongqing 400044

Received:2025-09-02 Revised:2025-09-12 Online:2025-10-20 Published:2025-10-11

摘要/Abstract

摘要： 目的探究甲基转移酶样蛋白3（methyltransferase-like 3, METTL3）通过m⁶A修饰调控整合素β3（ITGB3）表达促进巨核细胞分化的分子机制。方法本研究以人成巨核细胞白血病细胞系（MEG-01）为模型,结合20 nM佛波酯（phorbol myristate acetate, PMA）诱导分化体系。首先,通过蛋白质免疫印迹（western blot, WB）和实时荧光定量PCR（RT-qPCR）实验检测METTL3在MEG-01细胞中的表达。其次,利用慢病毒介导的shRNA（shMETTL3）构建METTL3敲低的MEG-01巨核细胞系。WB和RT-qPCR检测METTL3在MEG-01中的表达水平。流式细胞术检测巨核细胞表面分化成熟标志物CD41a和CD61的表达水平。转录组测序筛选METTL3的下游靶标分子,并通过RT-qPCR和WB检测其表达水平。继而,在过表达METTL3的细胞中,采用RT-qPCR和WB检测METTL3表达水平,流式检测CD41a、CD61。另外,在敲低METTL3的MEG01细胞中过表达METTL3,采用RT-qPCR和WB检测METTL3表达水平,流式检测CD41a、CD61。最后,生物信息学预测ITGB3 mRNA上的m⁶A结合位点。m⁶A-RIP联合Me-PCR实验检测m⁶A富集信号。结果 METTL3在MEG-01中mRNA及蛋白水平均有表达;成功构建METTL3敲低的MEG-01细胞系,METTL3 mRNA及蛋白表达水平均显著降低（P<0.001）;shMETTL3组MEG-01细胞表面巨核细胞分化成熟标志物CD41与CD61的表达水平较对照组显著下降（P<0.001）;转录组测序分析显示靶分子为ITGB3（CD61编码基因）,RT-qPCR与WB证实,shMETTL3组中ITGB3 mRNA及蛋白表达量显著降低（P<0.05）;METTL3过表达组CD41a和CD61显著上调;METTL3敲低后回补组CD41a和CD61显著恢复（P<0.01）;生物信息学预测及m⁶A-RIP联合Me-PCR实验证实：（1）ITGB3 mRNA的m⁶A富集程度显著高于对照GAPDH mRNA（P<0.001）;（2）shMETTL3组ITGB3 mRNA的m⁶A甲基化修饰水平明显降低（P<0.001）。结论 RNA甲基转移酶METTL3的下调导致ITGB3 mRNA的m⁶A甲基化修饰水平降低,进而抑制ITGB3表达,最终抑制巨核细胞分化成熟及血小板生成进程。

关键词: METTL3, m⁶A甲基化, 血小板生成, 巨核细胞分化, ITGB3, CD61

Abstract: Objective To investigate the molecular mechanism by which methyltransferase-like 3 (METTL3) promotes megakaryocyte differentiation via m⁶A modification-mediated regulation of integrin β3 (ITGB3) expression. Methods Human megakaryoblastic leukemia cells (MEG-01) treated with 20 nM phorbol myristate acetate (PMA) were used to establish a differentiation model. Baseline METTL3 expression in MEG-01 cells and hematopoietic stem cell (HSC) differentiation models was detected by Western blot (WB) and quantitative real-time PCR (RT-qPCR). Subsequently, lentivirus-mediated shRNA (shMETTL3) was used to transfect the MEG-01 megakaryocytic cell line. Knockdown efficiency was detected by WB and RT-qPCR. The expression levels of megakaryocytic surface differentiation markers CD41a and CD61 were measured by flow cytometry. Transcriptome sequencing was applied to identify downstream targets of METTL3, and their expression levels were validated by RT-qPCR and WB. Furthermore, in METTL3-overexpressing cells, the expression levels of METTL3 were detected by RT-qPCR and WB, and CD41a and CD61 were analyzed by flow cytometry. Additionally, METTL3 was re-expressed in METTL3-silenced MEG-01 cells, and its expression was assessed by RT-qPCR and WB, while the levels of CD41a and CD61 were detected by flow cytometry. Finally, bioinformatic prediction (SRAMP) was employed to identify potential m⁶A modification sites on ITGB3 mRNA, and m⁶A-RIP combined with Me-PCR was performed to examine the enrichment of m⁶A signals. Results METTL3 was expressed at mRNA and protein levels in MEG-01 cells and HSC-derived megakaryocytes.shMETTL3 significantly reduced METTL3 mRNA and protein (P<0.001). shMETTL3 decreased CD41a and CD61 surface expression versus the Scramble control (P<0.001). Transcriptomics identified ITGB3 (encoding CD61) as a key target; its mRNA and protein decreased in shMETTL3 cells (P<0.05). METTL3-OE upregulated CD41a and CD61; METTL3 rescue restored their expression (P<0.01). m⁶A-RIP/Me-PCR confirmed: (1) Higher m⁶A enrichment on ITGB3 versus GAPDH mRNA (P<0.001); (2) Reduced m⁶A modification on ITGB3 in shMETTL3 cells (P<0.001). Conclusion Downregulation of the RNA methyltransferase METTL3 reduces m⁶A modification on ITGB3 mRNA, thereby suppressing ITGB3 expression and impairing megakaryocyte differentiation and platelet production.

Key words: METTL3, m⁶A methylation, Platelet production, Megakaryocyte differentiation, ITGB3, CD61

中图分类号:

R331

侯晓宇, 夏文军, 陈睿驰, 孙登莲, 吴煌, 文爱清. METTL3介导ITGB3的m⁶A甲基化修饰促进巨核细胞分化^*[J]. 临床输血与检验, 2025, 27(5): 594-603.

HOU Xiaoyu, XIA Wenjun, CHEN Ruichi, SUN Denglian, WU Huang, WEN Aiqing. METTL3-mediated m⁶A Methylation Modification of ITGB3 Promotes Megakaryocyte Differentiation[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2025, 27(5): 594-603.

参考文献

[1] BOSCHER J,GUINARD I,ECKLY A,et al.Blood platelet formation at a glance[J]. J Cell Sci,2020,133(20).
[2] TYAGI T,JAIN K,GU S X,et al.A guide to molecular and functional investigations of platelets to bridge basic and clinical sciences[J]. Nat Cardiovasc Res,2022,1(3): 223-237.
[3] XU B C,YE X P,WEN Z Y,et al.Epigenetic regulation of megakaryopoiesis and platelet formation[J]. Haematologica, 2024,109(10):3125-3137.
[4] DAVENPORT P,LIU Z J,SOLA-VISNER M.Fetal vs adult megakaryopoiesis[J]. Blood,2022,139(22):3233-3244.
[5] PATEL S R.The biogenesis of platelets from megakaryocyte proplatelets[J]. J Clin Investig,2005,115(12): 3348-3354.
[6] MACHLUS K R,ITALIANO J E Jr. The incredible journey: From megakaryocyte development to platelet formation[J]. J Cell Biol,2013,201(6):785-796.
[7] 夏文军,卢尧,吴煌,等. m⁶A去甲基化酶FTO调控BCL2 mRNA稳定性与翻译效率促进血小板前体形成[J]. 陆军军医大学学报,2025,47(6):519-530.
[8] BESANCENOT R,ROOS-WEIL D,TONETTI C,et al.JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation[J]. Blood, 2014,124(13):2104-2115.
[9] CHOU F S,GRIESINGER A,WUNDERLICH M,et al.The thrombopoietin/MPL/Bcl-xL pathway is essential for survival and self-renewal in human preleukemia induced by AML1-ETO[J]. Blood,2012,120(4):709-719.
[10] HITCHCOCK I S,KAUSHANSKY K.Thrombopoietin from beginning to end[J]. Br J Haematol,2014,165(2): 259-268.
[11] HITZLER J K,CHEUNG J,LI Y,et al.GATA1 mutations in transient leukemia and acute megakaryoblastic leukemia of Down syndrome[J]. Blood,2003,101(11): 4301-4304.
[12] BEHRENS K,ALEXANDER W S.Cytokine control of megakaryopoiesis[J]. Growth Factors,2018,36(3/4): 89-103.
[13] MANCINI E,SANJUAN-PLA A,LUCIANI L,et al.FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors[J]. EMBO J, 2012,31(2):351-365.
[14] WANG C,TU Z W,CAI X W,et al.A critical role of RUNX1 in governing megakaryocyte-primed hematopoietic stem cell differentiation[J]. Blood Adv, 2023,7(11): 2590-2605.
[15] TIJSSEN M R,CVEJIC A,JOSHI A,et al.Genome-wide analysis of simultaneous GATA1/2,RUNX1,FLI1,and SCL binding in megakaryocytes identifies hematopoietic regulators[J]. Dev Cell,2011,20(5):597-609.
[16] BARTEL D P. microRNAs: genomics,biogenesis,mechanism,and function[J]. Cell,2004,116(2):281-297.
[17] GARZON R,PICHIORRI F,PALUMBO T,et al.microRNA fingerprints during human megakaryocytopoiesis[J]. Proc Natl Acad Sci USA,2006,103(13):5078-5083.
[18] BHATLEKAR S,MANNE B K,BASAK I,et al.miR-125a-5p regulates megakaryocyte proplatelet formation via the actin-bundling protein L-plastin[J]. Blood,2020,136(15):1760-1772.
[19] QU M Y,FANG F,ZOU X J,et al.miR-125b modulates megakaryocyte maturation by targeting the cell-cycle inhibitor p19(INK4D)[J]. Cell Death Dis,2016,7(10): e2430.
[20] WEISS C N,ITO K. microRNA-22 promotes megakaryocyte differentiation through repression of its target,GFI1[J]. Blood Adv,2019,3(1):33-46.
[21] CHEN X,YUAN Y X,ZHOU F,et al.RNA modification in normal hematopoiesis and hematologic malignancies[J]. MedComm,2024,5(11):e787.
[22] WU H,LU Y,SUN D L,et al.CircFUT8 promotes proplatelet formation by interacting with IGF₂BP₂ and stabilizing TNS1 mRNA in megakaryocytes[J]. Blood, 2025.
[23] 白秦,陈燕华,卢尧,等. 下调METTL3-LYN m6A-IGF₂BP₂通路抑制内皮细胞迁移[J]. 第三军医大学学报,2021, 43(9):845-851.
[24] 李秀丽,车舒平,曹荣祎. 血小板中lncRNA在结肠癌早期筛查中的应用价值[J]. 临床输血与检验,2025,27(2):200-206.
[25] SHI H H,CHAI P W,JIA R B,et al.Novel insight into the regulatory roles of diverse RNA modifications: re-defining the bridge between transcription and translation[J]. Mol Cancer,2020,19(1):78.
[26] JIANG X L,LIU B Y,NIE Z,et al.The role of m6A modification in the biological functions and diseases[J]. Signal Transduct Target Ther,2021,6(1):74.
[27] BAI Q,LU Y,CHEN Y H,et al.Endothelial METTL3 (methyltransferase-like 3) inhibits fibrinolysis by promoting PAI-1 (plasminogen activator inhibitor-1) expression through enhancing Jun proto-oncogene N6-methyladenosine modification[J]. Arterioscler Thromb Vasc Biol,2021,41(12):2877-2889.
[28] LV J H,ZHANG Y F,GAO S W,et al.Endothelial-specific m(6)a modulates mouse hematopoietic stem and progenitor cell development via Notch signaling[J]. Cell Res,2018,28(2):249-252.
[29] JIANG Z X,WANG Y N,LI Z Y,et al.The m6A mRNA demethylase FTO in granulosa cells retards FOS-dependent ovarian aging[J]. Cell Death Dis,2021,12(8): 744.
[30] LENCE T,AKHTAR J,BAYER M,et al.M(6)a modulates neuronal functions and sex determination in Drosophila[J]. Nature,2016,540(7632):242-247.
[31] YANKOVA E,BLACKABY W,ALBERTELLA M,et al.Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia[J]. Nature,2021,593(7860): 597-601.
[32] WANG Y,LI Y,TOTH J I,et al.N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells[J]. Nat Cell Biol,2014,16(2):191-198.
[33] ZHANG C X,CHEN Y S,SUN B F,et al.M(6)a modulates haematopoietic stem and progenitor cell specification[J]. Nature,2017,549(7671):273-276.
[34] WANG H,ZUO H N,LIU J,et al.Loss of YTHDF2-mediated m(6)A-dependent mRNA clearance facilitates hematopoietic stem cell regeneration[J]. Cell Res,2018, 28(10):1035-1038.
[35] CHENG Y M,LUO H Z,IZZO F,et al. M(6)a RNA methylation maintains hematopoietic stem cell identity and symmetric commitment[J]. Cell Rep,2019,28(7): 1703-1716.e6.
[36] GAO Y M,VASIC R,SONG Y B,et al. M(6)a modification prevents formation of endogenous double-stranded RNAs and deleterious innate immune responses during hematopoietic development[J]. Immunity,2020, 52(6):1007-1021.e8.
[37] LEE H,BAO S Y,QIAN Y Z,et al.Stage-specific requirement for Mettl3-dependent m(6)a mRNA methylation during haematopoietic stem cell differentiation[J]. Nat Cell Biol, 2019,21(6):700-709.
[38] LI D Y,PENG J,LI T T,et al.Itgb3-integrin-deficient mice may not be a sufficient model for patients with Glanzmann thrombasthenia[J]. Mol Med Rep,2021, 23(6):449.
[39] HUANG K,GAO J,DU J,et al.Generation and analysis of GATA2(w/eGFP) human ESCs reveal ITGB3/CD61 as a reliable marker for defining hemogenic endothelial cells during hematopoiesis[J]. Stem Cell Reports,2016, 7(5):854-868.
[40] KOMENO Y,UCHIYAMA T,KAWANO F,et al.Inherited macrothrombocytopenia due to a novel splice donor site mutation in ITGB3[J]. Ann Hematol,2023, 102(10):2947-2949.

METTL3介导ITGB3的m⁶A甲基化修饰促进巨核细胞分化^*

METTL3-mediated m⁶A Methylation Modification of ITGB3 Promotes Megakaryocyte Differentiation

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 6

编辑推荐

Metrics

本文评价

关于本刊

作者中心

在线期刊

访问统计

联系我们

[1]	岳伟, 杨玥, 周梅, 李津杞, 杨怡, 李彦欣, 钱宝华, 何啸, 顾海慧. hTERT在人诱导多能干细胞体外分化为巨核细胞中的作用^*[J]. 临床输血与检验, 2025, 27(5): 604-612.
[2]	周心蕙, 左斌, 王文, 何杨. ITP患者外周血脂质谱与促血小板生成药物疗效的相关性分析^*[J]. 临床输血与检验, 2025, 27(1): 115-120.
[3]	明静, 丁凯阳, 胡茂贵, 王馨辰. 阿伐曲泊帕联合rhTPO在恶性血液病自体造血干细胞移植中的临床疗效分析^*[J]. 临床输血与检验, 2024, 26(6): 812-817.
[4]	陈兰兰, 张燕华, 车进, 张嘉洪, 麻静敏, 李凤. 多次单采血小板对献血者血常规等指标的影响^*[J]. 临床输血与检验, 2021, 23(3): 332-336.
[5]	赵宏祥, 葛健民, 黄宏亮, 袁秀珍, 任素玲. 多次单采血小板献血者血清总蛋白、叶酸、血小板生成素变化的研究^*[J]. 临床输血与检验, 2018, 20(6): 604-607.
[6]	孙少勤. 重组人血小板生成素治疗血液肿瘤化疗所致血小板减少症疗效[J]. 临床输血与检验, 2018, 20(6): 632-634.