LncRNA-RIKEN在全麻药物引起的发育中大脑神经毒性中的作用及机制研究

General anaesthesia is usually considered to safely induce a reversible brain state allowing the performance of surgery under optimal conditions. An increasing number of clinical and experimental observations, however, suggest that anaesthetic drugs, especially when they are administered at the extremes of age, can trigger long-term morphological and functional alterations in the brain. To this end, mouse embryonic stem cells-drived neurons were used to simulate the neuronal differentiation system to systematically elucidate the effect of sevoflurane and propofol on neurodevelopment. We found that the long noncoding RNA RIKEN play very important role in general anaesthetics induced neurotoxicity, therefore, we will use mouse embryonic stem cells-drived neurons to detect the reduced expression of RIKEN induced by anesthetics. RIKEN, a competitive mRNA for miRNA-206-3p, facilitates miRNA-206-3p to down-regulate IGF-I expression, which can further inhibit the neuronal cell proliferation and neurite outgrowth, leading to impairment of learning and memory. In addition, fluorescent reporter gene and bioinformatics analysis were used to search for the transcription factor of RIKEN upstream in the promoter. This research provides a preventive target and theoretical basis for reducing the influence of anesthetics on neurodevelopment during long-term operation in infants and young children.

婴幼儿在手术中较长时间使用全身麻醉药物时，其对发育中大脑的远期影响是临床医生关注的焦点。回顾性研究发现有较长时间或者反复手术和麻醉药物暴露的儿童在青春期表现出认知功能损害的发生率较非暴露儿童要高。我们发现全麻药物抑制了神经干细胞分化，长链非编码RNA（RIKEN）在该进程中起着重要的作用。为此，我们拟利用神经干细胞向神经元分化体系，研究全身麻醉药物影响了发育中大脑的RIKEN的表达，RIKEN作为miRNA-206-3p的竞争性mRNA，影响miRNA-206-3p靶基因IGF-Ⅰ的表达，进而抑制神经干细胞分化，影响了后续分化的神经元轴突的形成和树突棘的成熟，导致远期学习和记忆能力损伤的下游机制；同时找寻作用于RIKEN核心启动子区并对其转录调控的上游转录因子并观察全麻药物对其上游转录因子的影响，从而为婴幼儿患者接受较长时间手术时降低全麻药物对神经发育的影响提供预防靶点和理论依据。

婴幼儿在手术中较长时间使用全身麻醉药物时，其对发育中大脑的远期影响是临床医生关注的焦点。回顾性研究发现有较长时间或者反复手术和麻醉药物暴露的儿童在青春期表现出认知功能损害的发生率较非暴露儿童要高。我们发现全麻药物抑制了神经干细胞分化，长链非编码RNA（RIKEN）在该进程中起着重要的作用。Rik-203在海马中的水平高于其他组织，并在神经分化过程中增加。七氟烷降低了Rik-203的水平。Rik-203的敲除降低了神经祖细胞的标志物Sox1和Nestin的mRNA水平，并减少了Sox1阳性细胞的数量。RNA pull-down显示，miR-101a-3p与Rik-203高度结合。最后，七氟烷、Rik-203的敲除和miR-101a-3p的过表达都降低了GSK-3β的水平。数据表明，Rik-203通过抑制miR-101a-3p降低GSK-3β水平的能力促进了神经分化，LncRNAs将作为麻醉神经毒性的机制。抑制miR-466l-3p可以恢复被Rik-203敲除后压抑的神经分化。脑源性神经营养因子（BDNF），被七氟烷下调，也被miR-466l-3p直接锁定。BDNF的过量表达恢复了被miR-466l-3p和Rik-203敲除抑制的神经分化。研究表明，七氟烷相关的LncRNARik-203通过抑制miR-466l-3p降低BDNF水平的能力促进神经分化。此外，研究发现Rik-201和Rik-203的抑制抑制了小鼠胚胎干细胞的神经分化。Rik-201和Rik-203作为竞争性内源性RNA（ceRNA），分别抑制miR-96和miR-467a-3p的功能，并调节Sox6的表达以进一步调节神经分化。敲除Rik-203和Rik-201会诱发高比例的大脑发育迟缓。此外，我们发现C/EBPβ有可能激活Rik-201和Rik-203的转录。这些发现确定了Rik-201和Rik-203在促进神经分化和进一步大脑发育中的功能作用，并阐明了潜在的miRNAs-Sox6相关的分子机制。