脊索动物文昌鱼(Branchiostoma belcheri)在动物免疫系统起源和演化研究中有特殊作用。目前，文昌鱼TLR通路研究取得了很大进展，然而相比于人类，miRNA在文昌鱼TLR通路调控研究明显不足。当前miRNA研究主要关注转录后/翻译水平负调控靶mRNA，却较少涉及核内miRNA作为激活子促进基因转录的研究。为揭示文昌鱼TLR免疫响应中的miRNA调控机制，本项目将在前期基础上，利用组学技术对参与TLR免疫响应的miRNA进行筛选，体内外验证miRNA抑制或激活靶基因的真实性，选取保守及文昌鱼特异的miRNA-靶基因研究miRNA在TLR免疫响应中反馈调节功能，揭示miRNA抑制和/或激活基因表达在文昌鱼TLR免疫响应中的作用机制。项目的完成不仅对阐明文昌鱼自身免疫调控机制具有重要科学意义，而且对脊椎动物免疫调控机制乃至其他物种中miRNA研究都具有重要的指导意义。

Amphioxus (Branchiostoma belcheri), as the representative head of the chordates in phylogenetic tree, provide valuable perspectives for investigating the origination and evolution of vertebrate immunity system. Till now, despite of great progress in amphioxus Toll-like receptor (TLR) signaling pathway, the regulating role of miRNA in the TLR related immune response of amphioxus remains elusive compared to that of mammal or human. Especially, the current miRNA researches mainly focus on the classic negative regulation of target mRNA by cytoplasmic miRNA at post-translational level (RNA interference, RNAi), while the new-discovered non-classic positive regulation of nuclear miRNA on target gene promoter at transcriptional level (RNA activation, RNAa) is attracting more and more attention. To further investigate the regulators involved in the innate immune-response to lipopolysaccharides (LPS) stimulation of amphioxus through TLR signaling based on preliminary results, this project proposal will first screen these immune-responsive miRNAs taking advantage of transcriptomic RNA-sequencing post nucleus and cytoplasm isolation, and validate the interaction between miRNAs and target genes via RNAi and/or RNAa using serial experiments in vitroand in vivo. Meanwhile the vertebrate conserved and/or amphioxus specific miRNA-target pairs were further confirmed and compared in human cells to study the mechanism of miRNA silencing and activating target genes in innate immune response of amphioxus TLR pathway. The accomplishment of this project would not only provide key insights into the innate immunity regulation of amphioxus, but also guide the future research on the vertebrate immunity and other systemic regulating mechanism by miRNAs.