血管衰老过程中NSUN2与METTL3/METTL14对衰老相关基因mRNA的协同甲基化以及血清m6A与m5C水平分析

RNA methylation is important for the gene regulation in cellular aging and vascular inflammation. However, the impact of RNA methylation in vascular aging and aging-related vascular disorders remain to be further studied. In previous study, we have found that: 1) NSUN2 and METTL3/METTL14 cooperatively methylate a lot of common target mRNAs encoding aging- or vascular disease-related factors for methylation; 2) the expression of NSUN2, METTL3, and METTL14 and vascular tissues could be induced by high-fat diet; 3) serum m5Cand m6A increased in high-fat diet mice but deceased in aged mice；4) The levels of m5C and m6A increased in Hyperlipidemia population, but decreased in aged population. Based on these findings, we set out to investigate the role of NSUN2- and METTL3/METTL14-mediated mRNA methylation in the regulation of aging-related genes and the mechanisms underlying these regulation, to investigate the impacts of NSUN2- and METTL3/METTL14-mediated mRNA methylation in vascular aging and aging-related vascular disorders, and to investigate whether serum RNA m5C and m6A levels could be used as a marker for aging and the aging-related vascular disorders. Our study will further reveal the mechanisms underlying aging and aging-related vascular disorders. Furthermore, these studies will be helpful for the pursuit of the current achievement in the area of RNA methylation toward clinical translational studies.

RNA 甲基化是细胞衰老与血管炎症的重要调控因素，但在血管衰老性病理过程中的意义尚待探讨。前期研究发现：1）NSUN2与METTL3/METTL14 (分别催化m5C与m6A甲基化) 协同甲基化许多衰老或血管病理相关mRNA； 2）高脂饮食诱导血管NSUN2、METTL3及METTL14表达；3）血清RNA m5C与m6A水平在高脂饮食小鼠升高但在衰老小鼠降低；4）血清RNA m5C与m6A水平在高脂血症人群有升高趋势，但在衰老人群则有降低趋势。本课题将进一步探讨NSUN2与METTL3/METTL14协同甲基化衰老相关基因mRNA并调控其表达的机制，探讨这些调控过程在血管衰老及相关病理过程中的意义，探讨血清RNA （或特定RNA）m5C与m6A水平是否可作为衰老及相关血管病理改变的标志。 这些研究将从新角度解析衰老与相关血管病理改变的机理，也将对RNA甲基化基础研究成果进行临床转化尝试。

衰老相关的炎症与代谢紊乱是包括血管在内的器官衰老的共性。衰老相关基因的转录调控已得到广泛深入研究，转录后调控正越来越受到重视。本课题在前期工作的基础上，进一步探讨了RNA甲基化与RNA结合蛋白介导的转录后基因调控、m5C与m6A甲基化之间的互作、RNA结合蛋白与RNA甲基化之间的互作在衰老或衰老相关病理改变过程中的作用与机制。结果显示，1）高脂与高胆固醇血症小鼠及人群血清RNA的m6A与m5C水平均明显升高；2）亚硫酸盐转化RNA的SOLiD™ 侧序分析与miCLIP分析得到了高脂与高胆固醇血症小鼠血清RNA甲基化的文库并分析了潜在标志物；3) RNA甲基化与衰老相关过程DNA损伤修复、造血干细胞再生、端粒稳态以及巨噬细胞极化相关; 4）血小板是衰老小鼠血清甲基化RNA的重要来源，血小板特异METTL3敲除则导致血小板源性炎症因子改变；5）RNA结合蛋白HuR可通过结合端粒酶非编码RNA TERC促进其m5C甲基化，进而维持周围细胞与造血干细胞端粒稳态；神经细胞则存在HuR与HuD/HuB竞争调控端粒酶活性（通过竞争结合TERC）的端粒稳态调控机制； 6）RNA结合蛋白HuR与Rhau可通过调控APOB、CYCS、UQCRB、NDUFB6、PLB、Nkx2-5 等基因mRNA加工、稳定性或翻译影响脂质代谢稳态与心肌重塑； 7）HuR可调控血小板PF4、IL-1β、CCL5、CD40L及SELP mRNA加工、稳定性或翻译，调控小肠上皮DGAT1、DGAT2及MGAT2的mRNA 半衰期或加工，影响血小板源性SASP及血管炎症，影响小肠脂质吸收；8）成功建立了可有效、特异筛选特定RNA分子之结合蛋白的方法，为转录后调控研究领域提供了新方法。在“Nature Communications”、“Nucleic Acids Research”、“Cardiovascular Research”、“Journal of Biological Chemistry”、"Cell Res."等杂志发表10篇论文（通讯/共同通讯及last author 5篇），另有4篇论文正在撰写投稿中。在从新角度探讨分子机制的同时，本课题也为RNA结合蛋白研究领域提供了新技术与手段..