JAK2可变剪接异构体调控周围神经再生的机制研究

The activation of pro-regenerative signaling pathway is crucial for peripheral nerve regeneration after injury. Identifying key molecules and regulation mode that involved in this process makes great contribution to the investigation of peripheral nerve injury repair. Alternative splicing (AS) is an important regulation mode of eukaryotic gene expression, which has been widely studied both in physiological and pathological processes. It has been reported that AS is of vital importance to the nervous system. However, the role of AS in peripheral nerve regeneration remains unclear. In this study, to identify the AS isoforms related to axon regeneration capacity, we analyzed the AS events during peripheral nerve injury repair by RNA-Seq. Moreover, we identified a key alternative splice isoform of JAK2, JAK2Δ14, as an important candidate in promoting axon regeneration. In addition to confirm its protein expression, we will also further determine the outcome of repairing rat sciatic nerve injury with JAK2Δ14 in vitro and in vivo and elucidate the mechanisms underlying the competitive regulation of JAK-STAT signaling pathway by JAK2Δ14 and JAK2 during this process to explore the potential of AS to promote peripheral nerve regeneration.

促轴突再生信号通路的激活是周围神经损伤再生过程的关键因素，深入探寻影响其激活的关键分子和调控模式有助于周围神经损伤修复的研究。作为真核基因表达调控的重要形式,可变剪接在神经系统中发挥了重要功能，而其在周围神经系统损伤修复过程中表达谱及功能至今鲜有报道。本立项通过RNA-Seq及生物信息学分析，筛选出与轴突再生相关的可变剪接异构体。以促轴突再生信号通路JAK-STAT中的JAK2Δ14为研究重点，确认该异构体的表达，通过体外和在体实验明确其在周围神经再生过程中的功能，并进一步探索JAK2Δ14与JAK2竞争性调控JAK-STAT信号通路的分子机制，探寻以JAK2Δ14为代表的可变剪接异构体促进神经再生的应用价值。

如何加速轴突生长是周围神经损伤修复领域的关键问题，寻找促神经再生因子对解决这一问题至关重要。作为真核基因表达调控的重要形式,可变剪接在神经系统中发挥了重要功能，而其在周围神经系统损伤修复过程中的表达谱及功能仍不清楚。本项目利用 RNA-Seq，获得了大鼠坐骨神经损伤后背根神经节（DRG）可变剪接表达谱，通过聚合酶链式反应（PCR）及 Sanger 测序等技术确认了Nrcam可变剪接异构体（Nrcam-S）的存在；利用体外原代神经元轴突生长模型及在体坐骨神经夹伤模型，确认了Nrcam-S促进坐骨神经再生的功能；找到了可能调控坐骨神经损伤后可变剪接事件的RNA结合蛋白CELF3和Rbfox2。由于环状RNA是一种特殊类型的可变剪接异构体，于是通过对已有数据进行进一步深入挖掘，获得了大鼠坐骨神经损伤后环状RNA表达谱。在DRG神经元中，利用核质分离、荧光原位杂交等技术发现了环状RNA circ-Spidr的富集；通过一系列体内外实验，证明了circ-Spidr能够促进DRG神经元轴突再生；并发现circ-Spidr部分通过激活PI3K-AKT信号通路从而实现对坐骨神经再生的诱导作用。在坐骨神经中，本项目寻找到特异性富集于施万细胞（SCs）的环状RNA circ-Ankib1，通过EdU增殖实验、荧光素酶、RNA结合蛋白免疫沉淀、实时定量PCR、western blot等技术阐明了circ-Ankib1 通过分子海绵形式吸附miR-423-5p、miR-485-5p和miR-666-3p，调节Cyp26b1的表达，从而控制SCs增殖，进而影响坐骨神经再生的功能和分子机制。综上，本项目不仅获得了大鼠坐骨神经损伤后，可变剪接及环状RNA的表达谱，为后续相关基础与临床研究提供了宝贵的数据资源；而且发现了Nrcam-S、circ-Spidr、circ-Ankib1等全新的轴突再生调节因子，为外周神经损伤修复提供了新的分子靶标。