miR-34a介导的SIRT1/FoxO1通路对团头鲂高糖代谢的调控机制

High dietary carbohydrate results in metabolic syndrome in fish and even cause nutritional disorders, which has attracted the attention of scientists worldwide, but the molecular regulation mechansim of carbohydrate metabolism in fish liver has not been elucidated clearly. In our previous studies, the liver microRNA expression libraries of Megalobrama amblycephala fed with controlled and high dietary carbohydrate has been established by high-throughput sequencing technology, and miR-34a was found significant correlation with high dietary carbohydrate metabolism. Therefore, Megalobrama amblycephala and miR-34a has been chosen as research object and target in this research. It is expected to determin the target gene of miR-34a throuth means of bioinformatics and luciferase reporter gene technology, reveal the time and space characteristics of the miRNA and target gene expression combined with the means of proteomics, immunofluorescence and co-immunoprecipitation technologies through miRNA overexpression and inhibition expression in vitro and in vivo. So that, the signal transduction mechanisms on high dietary carbohydrate metabolism mediated by miR-34a through SIRT1/FoxO1 pathway will be illuminated. Through this research, it will provide theoretical basis for molecular regulation mechanism on carbohydrate metabolism of microRNA, and biological targets and control strategies for nutritional metabolic disorders of Megalobrama amblycephala, Furthermore, it will enrich aquatic animal nutrition metabolism studies and achieve unification of academic research and application.

鱼类摄食高水平糖饲料后会出现代谢综合症，甚至引起营养性疾病，这一问题早已引起国内外科学家的关注，而鱼体肝脏糖代谢的分子调控机制一直未能确切阐明。申请者在前期工作中，通过高通量测序技术建立了高糖日粮饲喂前后团头鲂肝脏miRNA表达文库，分析发现miR-34a与肝脏高糖代谢显著相关。因此，本项目以团头鲂为研究对象，miR-34a为研究目标，通过生物信息学、荧光素酶报告基因技术确定miR-34a靶向基因；通过过表达与抑制目标miRNA，结合蛋白组学、免疫荧光和免疫共沉淀技术，从细胞与整体水平揭示miRNA与靶基因表达的时空特征，查明由miR-34a介导的SIRT1/FoxO1通路对团头鲂肝脏高糖代谢的信号传导机制。本项目研究结果将为揭示miRNA对团头鲂肝脏糖代谢调控的分子机制提供理论基础，为营养性糖代谢紊乱提供生物靶点和调控策略,丰富水生动物营养代谢调控研究内涵，实现学术意义与应用价值的统一。

团头鲂(Megalobrama amblycephala)，又称武昌鱼，2015年，在我国年产量达79.7万吨，是我国大宗淡水养殖鱼类之一。团头鲂对糖的耐受能力相对较弱，摄食高糖饲料不利于鱼类的生长和对饲料的利用。在前期的研究中发现，翘嘴红鲌、异育银鲫、团头鲂、青鱼长期摄食高水平碳水化合物日粮会出现糖代谢酶活性下降、血糖持续偏高、应激蛋白表达上升等高糖不耐受现象，甚至出现肝细胞肿大甚至病变等症状，引发营养代谢综合症。关于鱼类糖代谢功能调控的研究主要集中在肝脏胰岛素和INSR分泌、葡萄糖转运体数目、糖代谢酶活性等方面，而关于糖代谢调控信号传导机制研究却鲜见报道。近年，随着HiSeq技术和分子生物学技术的发展，microRNAs(miRNAs)在哺乳动物胰岛素分泌、血糖水平和糖代谢中的调控作用逐渐被确定。本研究以团头鲂为研究对象，通过HiSeq技术对团头鲂高糖代谢响应miRNAs的表达谱进行分析，筛选糖代谢关键miRNA(miR-34a)，结合生物信息学技术分析其靶基因调控网络，利用活体miRNA干扰技术从糖代谢酶活性调控信号传导途径层面进一步阐释miR-34a如何通过其关键靶向SIRT1/FoxO1通路调控糖代谢，为深入研究团头鲂糖代谢调控机制提供效应靶点。以上问题的解决有助于了解团头鲂糖代谢酶活性调控的分子机制，弥补鱼类营养相关研究领域的不足，更能够通过分子干预手段提高鱼类对饲料糖类利用能力，为团头鲂营养性代谢紊乱提供生物靶点和调控策略，实现该研究学术意义与应用价值的统一。