miR-486-3p在甲基苯丙胺诱导精神障碍中的分子作用机制研究

Researches have shown that miRNA plays an important role in both drug addiction and psychosis. Based on our previous studies, we have established the genome-wide miRNA expression profile in Han Chinese individules taking methamphetamine with and without psychosis, and identified that the expression of miR-486-3p in methamphetamine-associated psychosis (MAP) group was significantly decreased, which was confirmed by the subsequent experiments. However, the mechanism underlying these effects of miR-486-3p on MAP is still unknown. In the study, we will attempt to identify the change and distribution of miR-486-3p expression in NAc, VTA and PFC by using RT-PCR, FISH, and Fluorescence immunohistochemistry in the MAP mouse model. Meanwhile, we will establish models of over-expression/inhibition of miR-486-3p by using nuclei microinjection technology to identify the expression changes of miR-486-3p in mouse three nuclei (NAc, VTA and PFC) and illustrate the regulating role of miR-486-3p in the etiology of MAP. Furthermore, we will detect the changes of protein profiles from mouse three nuclei (NAc, VTA and PFC) by using iTRAQ to illuminate the regulatory target genes and relation pathways of miR-486-3p involved in MAP, and evaluate the genetic association relationship of the target genes and related pathways with MAP. Finally, we will establish Mir486-knockout mouse by using CRISPR/Cas9 to confirm the regulatory effect of miR-486-3p on MAP. The study, would help building a solid foundation for further elucidating the molecular mechanisms of MAP.

研究表明miRNA对药物成瘾和精神障碍均有重要作用。基于我们前期建立的甲基苯丙胺诱导精神障碍(MAP)人群样本miRNA表达谱，发现并验证了miR-486-3p在MAP中显著表达，但其对MAP的作用机制尚不明。本研究拟建立MAP小鼠模型，利用RT-PCR、荧光FISH和荧光免疫组化，明确miR-486-3p在3个核团(NAc及其投射相关的VTA和PFC)中的表达变化及分布；采用核团注射建立3个核团中miR-486-3p过表达/表达抑制模型，明确miR-486-3p在小鼠3个核团中的表达变化及对MAP的调控作用；利用iTRAQ检测小鼠3个核团中蛋白质谱表达改变，进一步阐明miR-486-3p参与MAP调控的靶基因及相关信号通路，并明确它们与MAP的遗传关联关系；最后通过CRISPR/Cas9构建Mir486敲除小鼠进一步验证miR-486-3p对MAP的作用，为阐明MAP的分子机制奠定基础

甲基苯丙胺诱导的精神分裂(MAP)，病程可以从短暂的中毒状态一直延续到长期的物质性诱发精神症或者类似于精神分裂症样的持续症状，MAP的神经生物学机制研究不但是法医学领域的重要课题，而且对维护社会和经济的健康发展，提高公民健康水平具有重要的促进作用。同时miRNA对药物成瘾和精神障碍均有重要作用。我们首先采用实时定量PCR分别检测METH诱导的MAP小鼠中脑腹侧背盖区（VTA）、伏隔核（NAc）、前额叶皮质（PFC）脑区中miR-486-3p的表达变化发现在VTA、NAc、PFC中miR-486-3p均高表达，并且在VTA中差异最大；第二步构建miR-486-3p过表达/表达抑制腺相关病毒（AAV），利用核团注射技术，将过表达/表达抑制AAV注射进入小鼠VTA，发现过表达miR-486-3p后小鼠更易引起MAP症状，表达抑制miR-486-3p后小鼠MAP症状改善，进一步确定miR-486-3p对MAP的分子作用机制；第三步通过数据库结合qRT-PCR方法筛选出靶基因并通过双荧光素报告基因法验证miR-486-3p对所筛选靶基因的调控作用，发现miR-486-3p可能通过调节ARID1B基因参与MAP调控调节。以上结果证明：1）VTA、NAc、PFC脑区中miR-486-3p调控MAP；2）在VTA中miR-486-3p可以通过ARID1B影响MAP。这些结果为MAP的防治提供了新思路。