miR-222调控DRG神经元参与神经再生的机制

The peripheral nervous system (PNS) has an intrinsic capacity to regenerate on its own. Deciphering the molecular mechanisms underlying nerve regeneration following rat sciatic nerve transection contributes to identifying the triggers and regulators of nerve regeneration. MicroRNAs (miRNAs) are a class of small, non-coding RNAs (～22 nucleotides) that negatively regulate gene expression post-transcriptionally either through translational inhibition or degradation of target mRNAs. miRNAs are proven to be involved in many biological processes. Dorsal root ganglia (DRGs) neurons spontaneously undergo robust neurite growth after axotomy. The roles of miRNAs in regulating the responses of DRG neurons to injury stimuli and activating their intrinsic growth capacity, however, are not fully understood. In preliminary experiments, we (1) screened miRNA profiling in couple with mRNA profiling in L4-6 DRGs following rat sciatic nerve transaction, and identified that 26 known miRNAs were differentially expressed over different time points; (2) integrated putative miRNA targets with differentially expressed mRNAs to yield potential targets, thus building the network for miRNAs and potential targets; (3) following global analysis, performed functional investigation by using in vitro cell model, and noted that miR-222 markedly promoted neurite outgrowth. In this project, we are going to search for the upstream factor that could regulate the miR-222 expression, and to explore the molecular mechanisms of miR-222 promoting regenerative outgrowth of DRG neurons by targeting PTEN and TIMP3. Moreover, we also plan to investigate the impact of miR-222 in an in vivo model by injecting it to tissue engineered nerve grafts to determine whether the exposure to miR-222 could enhance axon growth. We believe that the oncoming findings may aid to developing a new route to stimulate intrinsic neurite growth in peripheral nerve regeneration.

周围神经在损伤后能再生，分析损伤后能再生和部分恢复功能的坐骨神经有助于找到促进神经再生的关键启动和调控分子。microRNA (miRNA)是一类～22nt的内源性单链RNA分子，可诱导转录后基因沉默，在多种生物学过程中发挥重要调控作用。目前，microRNA在坐骨神经再生过程中背根神经元启动内在再生能力中的作用尚不明确。前期系统性分析了大鼠坐骨神经缺损后不同时间点背根神经节miRNA表达谱和mRNA表达谱；通过miRNA与基因的调控网络分析，结合功能筛选，得到网络中具有调控地位的miR-222及被调控的关键的靶基因(PTEN、TIMP3)。本课题将进一步明确调控miR-222表达的上游信号以及分析miR-222通过靶基因参与调控神经再生的分子机制；并用人工组织神经移植物桥接修复大鼠缺损坐骨神经，注射miR-222模拟物在体观察大鼠坐骨神经修复的效果，探讨miRNA作为新靶点的应用价值。

周围神经损伤后，由于神经元内在再生能力的激活以及施万细胞提供的再生微环境，周围神经能够实现成功再生，重新支配靶肌。L4-L6背根神经节（dorsal root ganglia，DRG）神经元是一种感觉神经元，其轴突分支组成了坐骨神经。在坐骨神经损伤后DRG神经元能维持存活和轴突再生，这个过程涉及众多转录因子的激活和再生相关基因的转录调控。.miRNA是一类长约22个核苷酸的非编码RNA，由一段具有发夹结构的单链RNA前体经Drosha酶和Dicer酶剪切后生成。miRNA可以靶向目标mRNA分子3’端非翻译区（3’-UTR），使mRNA分子降解或翻译受到抑制，在转录后水平负调控许多基因的表达。研究表明miRNA参与细胞的凋亡、增殖、迁移、分化、发育和代谢等过程，在许多生理和病理过程中起到非常重要的作用。本研究拟在坐骨神经损伤模型中，通过miRNA和mRNA芯片，筛选出调控背根神经节神经元表型，激活其内在再生能力的miRNA。.我们发现：（1）通过系统性分析坐骨神经损伤后L4-L6背根神经节miRNA的表达谱变化，结合生物信息学分析和功能筛选发现了miR-21和miR-222通过靶向TIMP3抑制背根神经节神经元的凋亡。另外，IL-6刺激背根神经节神经元可以上调miR-21的表达。（2）通过系统性分析坐骨神经损伤后L4-L6背根神经节miRNA的表达谱变化，结合生物信息学分析和功能筛选发现了miR-222通过靶向PTEN促进背根神经节神经元突起的生长。另外，c-Jun激活上调miR-222的表达，miR-222也可以通过PTEN调节CREB磷酸化。这有助我们从新的角度解释周围神经损伤和修复的机制，为将来的临床治疗提供新的思路。