MicroRNA-183-5p靶向Nrf2/HO-1轴调控脑出血后损伤修复机制的研究

Now people know little about the mechanism of the injury after ICH. It is very importment to find new genes and make sure how they work after ICH. MicroRNA is a kind of highly conserved RNA molecules in biology, involved in cell growth and differentiation, organism development and diseases. Our previous study has found that the expression of miRNA-183-5p is significantly different after ICH and it has some correlation with Nrf2 and HO-1. The mechanism of Nrf2/HO-1 after ICH is still unclear. We found that it may play an important role in brain injury and recovery via regulating its downstream signaling. Thus, we hypothesize that miRNA-183-5p may mediate brain injury and repair via regulating Nrf2/HO-1 signaling pathway after ICH. To confirm our hypothesize, we will use collagenase-induced ICH model in vivo, cell culture in vitro and gene sequencing, information biology, gene knockout, transfection, vector construction, combined with RT-PCR, Western blotting, immunofluorescence, Micro CT, MRI and some other experimental methods to elucidate the role of miRNA-183-5p after introcerebral hemorrhage. Based on our previous study, this study will determine the role of miRNA-183-5p in brain injury and neurologic functional recovery after ICH and its underlying mechanism involving inflammation, oxidative damage, neurogenesis and angiogenesis. The study will provide an effective treatment and therapeutic targets for ICH.

目前，人们对脑出血损伤的具体分子机制仍缺乏了解，揭示新基因在脑出血中的作用机制具有重要意义。近年研究发现MicroRNAs在细胞分化等过程中发挥重要作用。我们前期研究通过高通量测序筛选出脑出血后小鼠脑组织中miRNA-183-5p差异表达，通过生物信息学分析发现其与Nrf2和HO-1密切相关。基于此，我们提出研究假说：脑出血后诱导miRNA-183-5p发生改变，并通过对Nrf2/HO-1轴的调控介导脑出血后大脑损伤和神经功能恢复。我们将通过基因测序、生物信息学、基因敲除、转染、载体构建等多种分子生物学方法，从基因、蛋白、细胞、组织及动物整体水平多方面探讨，阐明miRNA-183-5p通过Nrf2/HO-1信号通路参与脑出血后调控的复杂分子机制。本研究基于前期研究基础，从炎症、氧化损伤、神经修复等角度深入探讨，为脑出血治疗及康复提供新的基因靶向治疗位点，具有重要的科学及临床意义。

HO-1广泛参与脑出血的继发性损伤过程。本课题通过对胶原酶诱导的脑出血后3天小鼠及假手术小鼠的脑组织进行microRNA测序，寻找差异性表达的microRNAs。综合生物信息学方法和miRNA测序结果，在表达差异最明显的15个miRNA中选择了以HO-1作为潜在结合靶点的miR-183-5p进行研究。然后，利用激活剂agomir和抑制剂antagomir对miR-183-5p进行调控，结合免疫荧光、Western blot、Elisa、qPCR、特殊染色等技术观察到miR-183-5p在脑出血早期具有抗神经炎症、氧化应激、减少脑血肿体积、减轻脑水肿、保护血脑屏障等作用。机制上，通过双荧光素酶报告实验证实了miR-183-5p和HO-1 mRNA具有靶向结合关系。在小鼠体内使用HO-1的化学抑制剂ZnPP、HO-1被下调的情况下，miR-183-5p在神经炎症，氧化应激的作用消失；同时体外实验利用BV2小鼠小胶质细胞，通过免疫荧光、Western blot、CCK-8等手段观察不同表达水平的miR-183-5p/HO-1对小胶质细胞活性的影响。上述的结果均证实了miR-183-5p的脑保护功能依赖于HO-1。最后，引入Nrf2基因敲除小鼠，Nrf2化学激动剂tBHQ，结合qPCR、Western blot的结果观察到Nrf2和miR-183-5p互相均存在负向调控关系，提示这可能是一种反馈调节，而并不是直接的相互作用。当HO-1被抑制后，上述反馈关系消失的结果以及miRNA预测不支持miR-183-5p与Nrf2结合的事实证明miR-183-5p与Nrf2之间存在着HO-1依赖性的负调节关系。本课题的结论是证实脑出血后小鼠脑组织miR-183-5p的表达明显降低。miR-183-5p通过抑制HO-1蛋白的表达来减轻氧化应激和神经炎症。此外， miR-183-5p与Nrf2之间存在HO-1依赖的负反馈调节关系。本课题研究为miR-183-5p/HO-1作为减轻脑出血继发神经炎症和氧化损伤潜在的靶点提供了理论支持。