颅脑创伤后冷休克蛋白RBM3调控星形胶质细胞外泌体源长链非编码RNA-MIR17HG促进神经突起再生的机制研究

Neurite regeneration after traumatic brain injury (TBI) is associated with neurological function improvement. Our previous study found that exosomes derived from injured astrocyte could promote neurite regeneration in mice after TBI. However, the underlying mechanism is still unknown. Our preliminary experiment found the level of lncRNA-MIR17HG was significantly elevated in the exosomes along with increased intracellular RBM3 expression after astrocyte injury. Moreover, RBM3 could directly combine with MIR17HG and increase its level in astrocyte-derived exosomes. Co-culture of injured neurons and exosomes derived from MIR17HG over-expression astrocyte could promote neurite regeneration. Bioinformatics prediction showed that MIR17HG could target miR-140 and regulate SIRT1 expression. Therefore, we propose a hypothesis: RBM3 regulates astrocyte-derived exosomal MIR17HG which promotes neurite regeneration through targeting miR-140 and regulating its downstream SIRT1/AKT pathway after TBI. This study investigates the role of RBM3 in promoting neurite regeneration through regulating astrocyte-derived exosomes after TBI and reveals the underlying mechanism of MIR17HG in regulating SIRT1 through targeting miR-140. Our study may provide a new molecular therapy target for TBI treatment.

颅脑创伤（TBI）后神经突起再生与神经功能恢复密切相关。我们预实验发现星形胶质细胞牵张损伤后，其外泌体可促进TBI小鼠神经突起再生，但机制不明。进一步实验发现：受损星形胶质细胞外泌体中长链非编码RNA-MIR17HG含量及胞内冷休克蛋白RBM3表达均显著升高，且RBM3可直接结合MIR17HG并增加其外泌体内含量，而上调外泌体内MIR17HG并递送损伤神经元可促进神经突起再生；生物信息学预测MIR17HG靶向miR-140调控SIRT1蛋白表达。故我们推测：TBI后星形胶质细胞通过上调RBM3增加其外泌体中MIR17HG含量，后者靶向miR-140激活神经元SIRT1通路促进神经突起再生。本课题拟从分子、细胞及动物水平阐明TBI后RBM3调控星形胶质细胞外泌体促进神经突起再生的作用，揭示MIR17HG靶向miR-140对SIRT1的调控机制，为TBI后神经功能康复治疗提供新思路及可靠靶点。

目的：颅脑创伤（TBI）后，过表达RBM3可促进损伤神经元修复，但机制尚不明确。本研究拟探讨RBM3通过调控星型胶质细胞来源外泌体LncRNA表达在TBI后神经修复中的作用机制。.方法：构建小鼠控制性脑损伤模型（CCI），尾静脉注射PKH26标记RBM3过表达星型胶质细胞来源及其对照组外泌体；ASO-NC及ASO-LncRNA XIST；Agomir NC及Agomir-20b-5p。高尔基染色检测神经元形态及棘突数量。二代测序检测RBM3过表达星型胶质细胞较对照组LncRNA表达差异，并通过RT-qPCR验证外泌体内LncRNA XIST含量差异。RIP实验验证RBM3是否直接结合LncRNA XIST。放线菌素实验检测RBM3过表达及对照组LncRNA XIST降解水平。RIP实验验证LncRNA XIST是否直接结合miR-20b-5p。行为学实验检测Agomir-20b-5p及对照组小鼠运动功能。WB检测miR-20b-5p mimic及对照组牵张损伤神经元细胞PTEN及p-AKT表达水平。Luciferase实验验证miR-20b-5p是否直接结合PTEN。RT-qPCR检测miR-20b-5p mimic及对照组BV2细胞内炎症因子表达水平。.结果：RBM3过表达星型胶质细胞来源外泌体显著增加CCI小鼠损伤周围区神经元树突数量，长度及棘突数量。二代测序结合RT-qPCR显示，RBM3过表达星型胶质细胞来源外泌体内LncRNA XIST含量较对照组显著下降。其机制为RBM3直接结合LncRNA XIST并促进其降解。CCI后尾静脉注射LncRNA XIST抑制剂ASO-LncRNA XIST或miR-20b-5p激动剂均可显著增加损伤周围区神经元树突数量，长度及棘突数量。其机制为抑制LncRNA XIST竞争性结合miR-20b-5p，促进miR-20b-5p吸附PTEN，进而激活PI3K-AKT通路，促进神经元自身再生能力。同时miR-20b-5p可被小胶质细胞摄取，并抑制M1型炎症因子含量。.结论：RBM3通过直接结合LncRNA XIST促进其降解，下调星型胶质细胞外泌体内LncRNA XIST含量，降低后者靶向吸附损伤神经元miR-20b-5p水平，进而下调PTEN蛋白表达，激活PI3K-AKT通路促进损伤神经元神经再生并改善小鼠运动功能。