本文選題：內含子　切入點：miR-932　出處：《東南大學》2016年博士論文　論文類型：學位論文

【摘要】：MicroRNA(miRNA)是一種長度在～22 nt可以調控蛋白質編碼基因表達的非編碼小分子RNA。過去二十多年,miRNA的研究取得了很大進展,但仍然有大部分miRNA功能未知,尤其是對內含子miRNA的功能研究知之甚少。神經系統(tǒng)是迄今最為復雜和精密的網絡系統(tǒng),由眾多神經元形成數量巨大的突觸連接而成,并受到miRNA的調控。本實驗室前期的研究發(fā)現Neuroligin2(dng2)參與突觸的形成與功能調節(jié)。有趣的是,我們發(fā)現其內含子區(qū)存在一段似miRNA的保守序列,分析發(fā)現其為miR-932�；诂F在的理論推測內含子miRNA應該可以直接反饋調節(jié)宿主基因表達,但是果蠅突觸粘附分子Neuroligin2(dnlg2)內含子區(qū)的miR-932功能未知。本文應用原位雜交及miRNA sensor實驗,顯示miR-932是一個胚胎、幼蟲及成蟲神經系統(tǒng)高表達的miRNA:qRT-PCR結果顯示在胚胎末期至蛹期dnlg2和miR-932 RNA水平呈此消彼長的趨勢。生物信息學分析預測miR-932在dnlg2的CDS編碼區(qū)有兩個作用位點。在體外(in vitro)雙熒光素酶報告基因系統(tǒng)及果蠅S2細胞轉染實驗發(fā)現,內含子miR-932可以通過直接靶向宿主dnlg2的編碼區(qū),下調dnlg2其RNA及蛋白表達水平。利用轉基因UAS-miR-932分別在神經、肌肉中過表達miR-932,引起不同程度的生長缺陷。以神經肌肉接頭NMJ為模型,研究顯示miR-932參與DNlg2介導的突觸發(fā)育與功能。與宿主基因dnlg2KO70突變體的表型(Bouton數目減少,GluRⅡB降低)相一致,miR-932過表達也引起GluRⅡB的減少。但是電生理檢測顯示,突觸前過表達miR-932后引起mEJP幅度增加、頻率不變;而在DNlg2 KO70突變體mEJP沒有變化,提示可能有其它潛在miR-932靶標分子參與這一過程。miR-932敲除(miR-932KO)及敲低(UAS-miR-932Sponge)實驗也均證實內含子miR-932可以下調宿主DNlg2表達量。上述結果顯示,內含子miR-932參與神經肌肉接頭處DNlg2介導的突觸后GluRⅡB降低及突觸分化和神經遞質傳遞。進一步分析全基因組中內含子區(qū)miRNA發(fā)現,果蠅中有44.4%內含子miRNA(48/108個)被預測可以靶向宿主基因的CDS區(qū);同時,Gene Ontology(GO)分析發(fā)現這些被其內含子miRNA作用的宿主基因,在功能上呈現一定富集(如發(fā)育過程的調節(jié)等)。類似的,我們在人類及線蟲中的分別找到60.6%(492/812個)、30.2%(19/63個)內含子miRNA可以靶向宿主基因的CDS區(qū)域。這提示我們的報導并非特例,而是一個廣泛存在的現象;這對于有效控制、維持宿主基因表達,尤其是神經相關突觸分子的穩(wěn)態(tài)具有重要的生物學意義。
[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，

本文編號：1644523