METTL3通过YTHDF2/AKT1/PPARγ调控人骨髓间充质干细胞成脂分化的机制研究

METTL3 regulates adipogenic differentiation of bone marrow MSCs via YTHDF2/AKT1/PPARγaxis

目的:探讨N^(6)-甲基腺苷(m^(6)A)甲基化酶甲基转移酶样蛋白3(METTL3)在体外调控人骨髓间充质干细胞(MSCs)向脂肪细胞分化的作用机制。方法:构建METTL3、AKT丝氨酸/苏氨酸激酶1(AKT1)、过氧化物酶体增殖物激活受体γ(PPARγ)和YTH m^(6)A RNA结合蛋白F2(YTHDF2)慢病毒载体,包装慢病毒并感染MSCs;采用成脂分化试剂盒诱导各组MSCs分化为脂肪细胞,利用油红O染色检测各组的脂肪细胞形成情况;采用分子克隆技术构建METTL3突变重组载体以证实METTL3的m^(6)A关键位点对靶基因的调控作用;利用放线菌素D作用于YTHDF2过表达的MSCs以探究阅读蛋白YTHDF2对AKT1 mRNA和蛋白表达的影响;采用RNA pull-down结合银染和Western blot实验检测可能发生甲基化修饰的片段与识别蛋白的结合情况。结果:m^(6)A甲基化酶METTL3以PPARγ/AKT1依赖的方式抑制骨髓MSCs的脂肪形成,并且以m^(6)A修饰阅读蛋白YTHDF2依赖的方式抑制AKT1的蛋白表达。YTHDF2可识别并结合发生m^(6)A修饰的AKT1编码序列(CDS),降低其表达水平,抑制MSCs脂肪形成过程。结论:m^(6)A甲基化酶METTL3通过YTHDF2/AKT1/PPARγ通路调控人骨髓MSCs成脂分化过程。本研究为从肿瘤微环境角度寻找急性髓系白血病治疗新靶点提供了理论依据。

AIM:To investigate the mechanism by which N^(6)-methyladenosine(m^(6)A)methylase methyltrans-ferase-like protein 3(METTL3)regulates the differentiation of human bone marrow mesenchymal stem cells(MSCs)into adipocytes in vitro.METHODS:Lentiviral vectors of METTL3,AKT serine/threonine kinase 1(AKT1),peroxisome pro-liferator-activated receptorγ(PPARγ)and YTH m^(6)A RNA binding protein F2(YTHDF2)were constructed to package lentiviral particles and used to infect MSCs.A human bone MSC adipogenic differentiation kit was used to induce the MSCs into adipocytes.Additionally,the adipocytes were stained by oil red O.The recombinant vector of METTL3 mutant was constructed using molecular cloning to confirm the regulatory effect of the key site of m^(6)A in METTL3 on the target genes.Actinomycin D was applied to MSCs with overexpression of YTHDF2 to evaluate the effect of YTHDF2 recognition on the mRNA and protein expression of AKT1.The RNA pull-down assay combined with silver staining and Western blot were used to detect the binding of potentially methylated fragments to recognized proteins.RESULTS:METTL3 inhibited the adipogenesis of MSCs in an AKT1/PPARγ-dependent manner,and mediated the protein expression of AKT1 in an m^(6)A-YTHDF2-dependent manner.YTHDF2 recognized and bound to coding sequence(CDS)of m^(6)A-AKT1,and reduced its expression,which inhibited the adipogenesis of MSCs.CONCLUSION:The m^(6)A methylase METTL3 regulates the adipo-genic differentiation of human bone marrow MSCs through YTHDF2/AKT1/PPARγ,providing a theoretical basis for the identification of new targets for acute myeloid leukemia treatment from the perspective of tumor microenvironment.