关于microRNAs通过靶向Nampt影响AMPK-NAD+/Sirt抗衰老信号通路的研究

Cellular senescence is the biological basis of age related diseases, so it is important to clarify its mechanism and identify novel targets for anti-aging therapeutics. Our previous results showed that AMPK’s anti-aging effect relates to its upregulation of NAD+ synthesis, particularly of the expression of rate-limiting enzyme of NAD+ synthase Nampt in mice. We also found that the expression of Nampt gene can be regulated at miRNA level. However, there is no reported intercrossing between miRNAs mechanism and the role of AMPK in upregulation of NAD+ synthesis. Given both the mechanisms of AMPK’s anti-aging effect and its regulation of Nampt are obscure, we believe that exploring the paricipation of miRNAs in these two processes is worthy for getting novel discoveries. In this project, we plan to utilize miRNA differential expression analysis, miRNA/Nampt 3’UTR recognition and docking analysis and other biological approaches to elucidate the role of specific miRNAs in AMPK’s effects on Nampt regulation, with cellular senescence and animal aging models. Our main goal is to identify miRNA molecules which participate and influence in the known AMPK-NAD+-SIRT1 pathway with a Nampt relevant way. Through this research, we hope to find a perspective orentation for digging the mechanism of aging and AMPK’s anti-aging effect . We also hope to get cues for finding novel molecular targets for aging and age-related diseases.

衰老是老年性疾患发生的生物学基础，故阐明其发生机制并找到阻遏靶点有重要意义。 我们的前期工作证实AMPK的抗衰老作用与其上调NAD+合成和NAD+合成限速酶NAMPT的表达有关。虽然已知众多基因表达受microRNAs(miRNAs)机制调控，但这种机制是否影响AMPK上调NAD合成和抗衰老尚不清楚。鉴于AMPK抗衰老和Nampt表达调控机制均不清楚，我们认为探讨两者之间由miRNA介导的联系可能带来全新发现。本项目拟利用细胞和动物衰老模型，通过miRNA差异表达分析，miRNA与Nampt3'UTR序列的识别和对接检测及其它生物学方法，解析miRNA影响AMPK上调Nampt基因表达的作用和机制。主要目标是在已知的AMPK-NAD+/SIRT1抗衰老信号通路中，发现新的分子节点和调控关系，揭示AMPK通过代谢途径阻遏衰老的新机制，并为研发抵御衰老及老年病策略提供新的分子靶标。

本项目已完成计划书所列工作内容，包括用组学方法筛选差异miRNA，并确定NAMPT靶向miRNA, 然后证明该miRNA对NAD/SIRT通路的影响， 解析AMPK如何调节该miRNA表达。各部分研究进展符合项目计划书所列年度计划。至目前已达成主体研究目标，即证明和阐释了miR-146a分子通过靶向NAMPT影响AMPK-SIRT抗衰老上下游信号通路，该工作已被Signal Transduction and Targeted Therapy期刊正式接受（中科院一区，IF=18.187)。相关发现加深和丰富了对AMPK-SIRT抗衰老通路的认识，明确了miR-146a在其中的作用，为进一步开展对该通路的调控和干预提供了新的理论和实验基础。 除了有创新性的研究发现和发表多篇标注的SCI论文，项目还培养了具备较好科研能力的硕士和博士研究生。相关研究内容有进一步拓展和深化前景，且在继续进行。..本项目的研究目标是探究和阐明参与AMPK-NAD/SIRT抗衰老信号通路调控的miRNA分子及其上下游信号通路，重点关注调控NAD合成关键酶NAMPT表达的miRNA分子及信号通路。按照设计的研究内容和技术路线，我们首先通过小RNA测序和生物信息学分析法，筛选到在衰老细胞中高表达并受AMPK负性调控的差异miRNA，而后通过多种细胞和分子生物学方法，证明了这些差异miRNA之一的miR-146a能直接靶向NAMPT并抑制其表达，还解析了miR-146a靶向NAMPT的位点，在体内外水平获取了miR-146a降低NAD/SIRT1活性，促进衰老的证据。 除了证明所连接的下游通路，我们更解析了AMPK对miR-146a表达的负性调控机制，发现AMPK激活通过抑制NFKB活性进而降低miR-146a的转录，揭示了新的miR-146a上游通路。这些发现拓展和充实了AMPK-SIRT抗衰老通路中的分子级联关系，为AMPK活化的抗衰老作用提供了新的阐释， 也为将miR-146a和NAMPT作为新的衰老干预靶点提供了新的研究基础。..此外，本项目还发现并解析了另一种受AMPK调控的衰老相关miRNA。该工作已大部完成，论文在撰写之中。该发现的特点是认识到一种新的抗衰老miRNA。这种miRNA的表达被AMPK上调，其靶点之一是调节mTOR活性和细胞自噬的蛋白，也与AMPK-SIRT抗衰老通路有关。