DataSheet1_N6-Methyladenosine Methyltransferase METTL3 Promotes Angiogenesis and Atherosclerosis by Upregulating the JAK2/STAT3 Pathway via m6A Reader IGF2BP1.docx

Atherosclerosis (AS) is a life-threatening vascular disease. RNA N6-methyladenosine (m6A) modification level is dysregulated in multiple pathophysiologic processes including AS. In this text, the roles and molecular mechanisms of m6A writer METTL3 in AS progression were explored in vitro and in vivo. In the present study, cell proliferative, migratory, and tube formation capacities were assessed through CCK-8, Transwell migration, and tube formation assays, respectively. RNA m6A level was examined through a commercial kit. RNA and protein levels of genes were measured through RT-qPCR and western blot assays, respectively. VEGF secretion level was tested through ELISA assay. JAK2 mRNA stability was detected through actinomycin D assay. The relationship of METTL3, IGF2BP1, and JAK2 was investigated through bioinformatics analysis, MeRIP, RIP, RNA pull-down, and luciferase reporter assays. An AS mouse model was established to examine the effect of METTL3 knockdown on AS development in vivo. The angiogenetic activity was examined through chick chorioallantoic membrane assay in vivo. The results showed that METTL3 was highly expressed in ox-LDL-induced dysregulated HUVECs. METTL3 knockdown inhibited cell proliferation, migration, tube formation, and VEGF expression/secretion in ox-LDL-treated HUVECs, hampered AS process in vivo, and prevented in vivo angiogenesis of developing embryos. METTL3 positively regulated JAK2 expression and JAK2/STAT3 pathway in an m6A dependent manner in HUVECs. IGF2BP1 positively regulated JAK2 expression through directly binding to an m6A site within JAK2 mRNA in HUVECs. METTL3 knockdown weakened the interaction of JAK2 and IGF2BP1. METTL3 exerted its functions through JAK2/STAT3 pathway. In conclusion, METTL3 knockdown prevented AS progression by inhibiting JAK2/STAT3 pathway via IGF2BP1.

动脉粥样硬化（AS）是一种危及生命的血管疾病。RNA N6-甲基腺苷（m6A）修饰水平在包括AS在内的多种病理生理过程中失调。在本研究中，通过体外和体内实验，探讨了m6A写者METTL3在AS进展中的功能和分子机制。本研究中，分别通过CCK-8、Transwell迁移和管形成实验评估了细胞的增殖、迁移和管形成能力。通过商业化试剂盒检测RNA m6A水平。通过RT-qPCR和蛋白质印迹实验分别测定基因的RNA和蛋白质水平。通过ELISA实验检测VEGF分泌水平。通过放线菌素D实验检测JAK2 mRNA的稳定性。通过生物信息学分析、MeRIP、RIP、RNA pull-down和荧光素酶报告基因实验研究METTL3、IGF2BP1和JAK2之间的关系。建立了AS小鼠模型，以检查METTL3敲低对体内AS发展的影响。通过体内鸡胚绒毛尿囊膜实验检测血管生成活性。结果显示，METTL3在ox-LDL诱导的失调的HUVECs中高表达。METTL3敲低抑制了ox-LDL处理的HUVECs中的细胞增殖、迁移、管形成和VEGF表达/分泌，阻碍了体内AS过程，并防止了发育胚胎的体内血管生成。METTL3以m6A依赖的方式在HUVECs中正向调节JAK2表达和JAK2/STAT3通路。IGF2BP1通过直接结合到JAK2 mRNA中的m6A位点，在HUVECs中正向调节JAK2表达。METTL3敲低削弱了JAK2和IGF2BP1之间的相互作用。METTL3通过JAK2/STAT3通路发挥其功能。总之，METTL3敲低通过抑制JAK2/STAT3通路，经IGF2BP1介导，防止了AS的进展。