甲基转移酶样3对巨核细胞系花生四烯酸代谢通路的影响和机制  

Impact and mechanism of methyltransferase-like 3 on regulation of arachidonic acid metabolism in megakaryocytic lines

作　　者：张嘉祺 刘雯雯[1] 王茜婷[1] 陈夏欢[1] 刘梅林[1] Zhang Jiaqi;Liu Wenwen;Wang Xiting;Chen Xiahuan;Liu Meilin(Department of Geriatrics,Peking University First Hospital,Beijing 100034,China)

机构地区：[1]北京大学第一医院老年病内科,100034

出　　处：《中国心血管杂志》2025年第1期75-85,共11页Chinese Journal of Cardiovascular Medicine

基　　金：北大百度基金资助项目(2019BD019);北京大学第一医院高质量临床研究专项(2023HQ04)。

摘　　要：目的探讨甲基转移酶样3(METTL3)对人成巨核细胞白血病细胞(MEG-01)花生四烯酸(AA)代谢通路的影响和相关机制。方法构建METTL3差异表达的MEG-01,分为正常对照(Blank)组、METTL3过表达(OE)组和过表达阴性对照(OE-NC)组、METTL3敲低(KD)组和敲低阴性对照(KD-NC)组。采用定量聚合酶链反应(qPCR)法和免疫印迹(Western blot)法检测各组METTL3、YTH结构域家族蛋白(YTHDF1、YTHDF2)以及参与AA代谢的关键基因前列腺素内过氧化物合酶1/环氧合酶-1(PTGS1/COX-1)、前列腺素内过氧化物合酶2/环氧合酶-2(PTGS2/COX-2)、血栓素A合酶1(TBXAS1)、前列腺素I_(2)合酶(PTGIS)和前列腺素I_(2)受体(PTGIR)的表达量变化。采用酶联免疫吸附分析(ELISA)法测定细胞培养上清中AA代谢产物前列腺素H_(2)(PGH_(2))和血栓素B_(2)(TXB_(2))的水平。采用蛋白稳定性实验和RNA结合蛋白质免疫沉淀(RIP)实验验证YTHDF1与COX-1 mRNA的结合情况。结果qPCR和Western blot检测显示,OE组YTHDF1、PTGS1/COX-1的mRNA和蛋白水平较OE-NC组升高,KD组较KD-NC组降低(均为P<0.05)。ELISA测定显示,细胞培养上清中OE组PGH_(2)、TXB_(2)的含量高于OE-NC组,KD组TXB_(2)的含量低于KD-NC组(均为P<0.05)。蛋白稳定性实验和RIP实验证实,METTL3通过YTHDF1参与COX-1 mRNA的翻译调控。结论METTL3可能通过促进N6-甲基腺苷(m6A)修饰介导的COX-1 mRNA翻译效率,调控巨核细胞系的AA代谢通路。Objective To investigate the role and underlying mechanisms of methyltransferase-like 3(METTL3)on regulation of arachidonic acid(AA)metabolism pathway in the human megakaryocytic leukemia cell line(MEG-01).Methods MEG-01 cells with altered METTL3 expression were categorized into control,overexpression(OE),overexpression negative control(OE-NC),knockdown(KD),and knockdown negative control(KD-NC)groups.The expression levels of METTL3,YTH domain family proteins(YTHDF1,YTHDF2),and key genes involved in AA metabolism--prostaglandin-endoperoxide synthase 1/cyclooxygenase-1(PTGS1/COX-1),prostaglandin-endoperoxide synthase 2/cyclooxygenase-2(PTGS2/COX-2),thromboxane A synthase 1(TBXAS1),prostacyclin synthase(PTGIS),and prostacyclin receptor(PTGIR)--were assessed using quantitative polymerase chain reaction(qPCR)and Western blot.The concentrations of AA metabolites,prostaglandin H2(PGH2),and thromboxane B2(TXB2)in the cell culture supernatants were quantified using enzyme-linked immunosorbent assay(ELISA).Protein stability assays and RNA-binding protein immunoprecipitation(RIP)experiments were performed to verify the binding between YTHDF1 and COX-1 mRNA.Results In the OE group,the levels of YTHDF1,PTGS1(COX-1)mRNA,and protein were significantly higher than those in the OE-NC group;while in the KD group,these levels were significantly lower than those in the KD-NC group(all P<0.05).The contents of PGH2 and TXB2 in the culture supernatant of the OE group were higher than those in the OE-NC group;conversely,the content of TXB2 was lower in the KD group compared to the KD-NC group(all P<0.05).Protein stability and RIP experiments demonstrated that METTL3 modulates the translation of COX-1 mRNA through YTHDF1.Conclusions METTL3 may regulate AA metabolism in the megakaryocytic lines by promoting the N6-methyladenosine(m6A)-mediated translational efficiency of COX-1 mRNA.

关 键 词：甲基转移酶样3 N6-甲基腺苷甲基化 MEG-01细胞 花生四烯酸 心血管疾病 

分 类 号：R54[医药卫生—心血管疾病]