本文選題：內(nèi)含子　切入點(diǎn)：miR-932　出處：《東南大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：MicroRNA(miRNA)是一種長(zhǎng)度在～22 nt可以調(diào)控蛋白質(zhì)編碼基因表達(dá)的非編碼小分子RNA。過(guò)去二十多年,miRNA的研究取得了很大進(jìn)展,但仍然有大部分miRNA功能未知,尤其是對(duì)內(nèi)含子miRNA的功能研究知之甚少。神經(jīng)系統(tǒng)是迄今最為復(fù)雜和精密的網(wǎng)絡(luò)系統(tǒng),由眾多神經(jīng)元形成數(shù)量巨大的突觸連接而成,并受到miRNA的調(diào)控。本實(shí)驗(yàn)室前期的研究發(fā)現(xiàn)Neuroligin2(dng2)參與突觸的形成與功能調(diào)節(jié)。有趣的是,我們發(fā)現(xiàn)其內(nèi)含子區(qū)存在一段似miRNA的保守序列,分析發(fā)現(xiàn)其為miR-932�；诂F(xiàn)在的理論推測(cè)內(nèi)含子miRNA應(yīng)該可以直接反饋調(diào)節(jié)宿主基因表達(dá),但是果蠅突觸粘附分子Neuroligin2(dnlg2)內(nèi)含子區(qū)的miR-932功能未知。本文應(yīng)用原位雜交及miRNA sensor實(shí)驗(yàn),顯示miR-932是一個(gè)胚胎、幼蟲(chóng)及成蟲(chóng)神經(jīng)系統(tǒng)高表達(dá)的miRNA:qRT-PCR結(jié)果顯示在胚胎末期至蛹期dnlg2和miR-932 RNA水平呈此消彼長(zhǎng)的趨勢(shì)。生物信息學(xué)分析預(yù)測(cè)miR-932在dnlg2的CDS編碼區(qū)有兩個(gè)作用位點(diǎn)。在體外(in vitro)雙熒光素酶報(bào)告基因系統(tǒng)及果蠅S2細(xì)胞轉(zhuǎn)染實(shí)驗(yàn)發(fā)現(xiàn),內(nèi)含子miR-932可以通過(guò)直接靶向宿主dnlg2的編碼區(qū),下調(diào)dnlg2其RNA及蛋白表達(dá)水平。利用轉(zhuǎn)基因UAS-miR-932分別在神經(jīng)、肌肉中過(guò)表達(dá)miR-932,引起不同程度的生長(zhǎng)缺陷。以神經(jīng)肌肉接頭NMJ為模型,研究顯示miR-932參與DNlg2介導(dǎo)的突觸發(fā)育與功能。與宿主基因dnlg2KO70突變體的表型(Bouton數(shù)目減少,GluRⅡB降低)相一致,miR-932過(guò)表達(dá)也引起GluRⅡB的減少。但是電生理檢測(cè)顯示,突觸前過(guò)表達(dá)miR-932后引起mEJP幅度增加、頻率不變;而在DNlg2 KO70突變體mEJP沒(méi)有變化,提示可能有其它潛在miR-932靶標(biāo)分子參與這一過(guò)程。miR-932敲除(miR-932KO)及敲低(UAS-miR-932Sponge)實(shí)驗(yàn)也均證實(shí)內(nèi)含子miR-932可以下調(diào)宿主DNlg2表達(dá)量。上述結(jié)果顯示,內(nèi)含子miR-932參與神經(jīng)肌肉接頭處DNlg2介導(dǎo)的突觸后GluRⅡB降低及突觸分化和神經(jīng)遞質(zhì)傳遞。進(jìn)一步分析全基因組中內(nèi)含子區(qū)miRNA發(fā)現(xiàn),果蠅中有44.4%內(nèi)含子miRNA(48/108個(gè))被預(yù)測(cè)可以靶向宿主基因的CDS區(qū);同時(shí),Gene Ontology(GO)分析發(fā)現(xiàn)這些被其內(nèi)含子miRNA作用的宿主基因,在功能上呈現(xiàn)一定富集(如發(fā)育過(guò)程的調(diào)節(jié)等)。類似的,我們?cè)谌祟惣熬�蟲(chóng)中的分別找到60.6%(492/812個(gè))、30.2%(19/63個(gè))內(nèi)含子miRNA可以靶向宿主基因的CDS區(qū)域。這提示我們的報(bào)導(dǎo)并非特例,而是一個(gè)廣泛存在的現(xiàn)象;這對(duì)于有效控制、維持宿主基因表達(dá),尤其是神經(jīng)相關(guān)突觸分子的穩(wěn)態(tài)具有重要的生物學(xué)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(hào)：1644523