果蝇突触粘附分子Neuroligin2基因内含子区的miR-932功能研究

发布时间：2018-03-21 15:48

  本文选题：内含子　切入点：miR-932　出处：《东南大学》2016年博士论文　论文类型：学位论文

【摘要】：MicroRNA(miRNA)是一种长度在～22 nt可以调控蛋白质编码基因表达的非编码小分子RNA。过去二十多年,miRNA的研究取得了很大进展,但仍然有大部分miRNA功能未知,尤其是对内含子miRNA的功能研究知之甚少。神经系统是迄今最为复杂和精密的网络系统,由众多神经元形成数量巨大的突触连接而成,并受到miRNA的调控。本实验室前期的研究发现Neuroligin2(dng2)参与突触的形成与功能调节。有趣的是,我们发现其内含子区存在一段似miRNA的保守序列,分析发现其为miR-932。基于现在的理论推测内含子miRNA应该可以直接反馈调节宿主基因表达,但是果蝇突触粘附分子Neuroligin2(dnlg2)内含子区的miR-932功能未知。本文应用原位杂交及miRNA sensor实验,显示miR-932是一个胚胎、幼虫及成虫神经系统高表达的miRNA:qRT-PCR结果显示在胚胎末期至蛹期dnlg2和miR-932 RNA水平呈此消彼长的趋势。生物信息学分析预测miR-932在dnlg2的CDS编码区有两个作用位点。在体外(in vitro)双荧光素酶报告基因系统及果蝇S2细胞转染实验发现,内含子miR-932可以通过直接靶向宿主dnlg2的编码区,下调dnlg2其RNA及蛋白表达水平。利用转基因UAS-miR-932分别在神经、肌肉中过表达miR-932,引起不同程度的生长缺陷。以神经肌肉接头NMJ为模型,研究显示miR-932参与DNlg2介导的突触发育与功能。与宿主基因dnlg2KO70突变体的表型(Bouton数目减少,GluRⅡB降低)相一致,miR-932过表达也引起GluRⅡB的减少。但是电生理检测显示,突触前过表达miR-932后引起mEJP幅度增加、频率不变;而在DNlg2 KO70突变体mEJP没有变化,提示可能有其它潜在miR-932靶标分子参与这一过程。miR-932敲除(miR-932KO)及敲低(UAS-miR-932Sponge)实验也均证实内含子miR-932可以下调宿主DNlg2表达量。上述结果显示,内含子miR-932参与神经肌肉接头处DNlg2介导的突触后GluRⅡB降低及突触分化和神经递质传递。进一步分析全基因组中内含子区miRNA发现,果蝇中有44.4%内含子miRNA(48/108个)被预测可以靶向宿主基因的CDS区;同时,Gene Ontology(GO)分析发现这些被其内含子miRNA作用的宿主基因,在功能上呈现一定富集(如发育过程的调节等)。类似的,我们在人类及线虫中的分别找到60.6%(492/812个)、30.2%(19/63个)内含子miRNA可以靶向宿主基因的CDS区域。这提示我们的报导并非特例,而是一个广泛存在的现象;这对于有效控制、维持宿主基因表达,尤其是神经相关突触分子的稳态具有重要的生物学意义。
[Abstract]:MicroRNAs miRNAs are a non-coding small molecule that can regulate the expression of protein-encoded genes at 22 NT. In the past 20 years, great progress has been made in the study of miRNAs, but most of the miRNA functions are still unknown. In particular, little is known about the function of intron miRNA. The nervous system is by far the most complex and sophisticated network system, formed by a large number of neurons formed by a large number of synaptic connections. Neuroligin2dng2) was found to be involved in synapse formation and function regulation. Interestingly, we found that there is a conserved sequence similar to miRNA in the intron region of Neuroligin2dng2. It was found to be miR-932. Based on the present theory, it was inferred that intron miRNA could regulate host gene expression directly, but the miR-932 function of intron region of synaptic adhesion molecule Neuroligin2 dnlg2 in Drosophila melanogaster was unknown. In situ hybridization and miRNA sensor experiments were used in this study. Shows that miR-932 is an embryo, The miRNA:qRT-PCR results of high expression in larva and adult nervous system showed that the levels of dnlg2 and miR-932 RNA increased from the end of embryo to the pupa. Bioinformatics analysis predicted that miR-932 had two action sites in the CDS coding region of dnlg2. Vitro-double luciferase reporter gene system and fruit fly S2 cell transfection assay. Intron miR-932 can down-regulate the expression of RNA and protein in dnlg2 by directly targeting the coding region of host dnlg2. The expression of miR-932 is overexpressed in nerve and muscle by transgenic UAS-miR-932, which leads to different degrees of growth defects. NMJ of neuromuscular junction is used as a model. It has been shown that miR-932 is involved in the development and function of DNlg2 mediated synapses. In line with the decrease in the number of host gene dnlg2KO70 mutants and the decrease of GluR 鈪，

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