miR-124通过调控RhoG来调控嗅球颗粒细胞发育

发布时间：2018-03-30 21:05

本文选题：嗅球　切入点：MicroRNAs　出处：《浙江大学》2016年博士论文

【摘要】：嗅觉系统是属于边缘系统(limbic system)的一部分,由嗅觉上皮,主嗅球和嗅皮质组成。嗅觉在动物寻找食物,识别危险,寻找配偶和社会交往具有重要的意义。成体室下区(SVZ)是哺乳动物成体大脑神经元发生区域之一。从SVZ神经母细胞(neuroblast)沿嘴侧迁移流(RMS)迁移到OB和分化成颗粒细胞(GCs)和球周细胞(PGs),然后整合到嗅球的突触环路。颗粒细胞属于抑制性神经元其数量占嗅球中间神经元的95%。他们的包体在颗粒细胞层(GCL)中生长几个短基底树突,而顶树突横跨僧帽细胞层(MCL),延申到外丛状层(EPL)。microRNA属于Small ncRNAs,是单链的短RNA,长度在18-25个核苷酸分子左右。microRNA是转录后基因表达的主要调节分子。microRNA以序列互补的方式与特异靶mRNA结合,通过降解靶基因mRNA或抑制其蛋白翻译调控靶基因的表达。microRNA在神经元发育、脑退行性疾病中发挥重要作用。miR-124是一种脑特定的miRNA,在小鼠大脑的分化和成熟的神经元中占所有大脑的miRNA的25-48%。miR-124其表达的水平,在发育的小鼠的中枢神经系统(CNS)中是逐渐增加的。miR-124决定成体室下区(SVZ)神经元前体的命运,并调节成年神经元在SVZ的再生。miR-124重要功能是调控神经元发育以及在神经退行性疾病中发挥重要作用。小G蛋白(Small G Protein),因分子量只有20～30KD而得名,是一类位于膜内侧的偶联蛋白,通过与膜受体胞浆区结合,将膜受体与配体结合的刺激与下游的效应分子偶联起来。RhoGTPases家族通过调控肌动蛋白细胞骨架,在突触可塑性神经元的树突棘的形态发生中发挥重要的作用。通过miRNA芯片方法揭示了嗅球的miRNA表达谱,发现miR-124是嗅球中最高表达的miRNA。通过靶基因预测以及功能网络富集,我们了解到miR-124参与调控细胞骨架重要功能。通过构建miR-124过表达和抑制慢病毒载体,并通过脑立体定位注射进小鼠的RMS,我们证明了 miR-124在小鼠嗅球颗粒细胞发育后期调控树突形态发育以及树突棘密度。在miR-124调控细胞骨架靶基因富集组分中我们发现小G蛋白RhoG可能具有重要作用。通过构建了 RhoG的过表达载体,RhoG的组成性激活突变,RhoG的组成性失活载体以及RhoG的shRNA慢病毒载体。通过脑立体定位注射到小鼠的RMS,我们观察RhoG是否能促进嗅球颗粒细胞树突形态发育以及树突棘密度。结果显示RhoG是通过下游Rac1的激活以及PAK和confilind的磷酸化发挥作用的。RhoG的表达模式与miR-124很相似。这种看似矛盾但是可以理解为RhoG有可能参与调控迁移作用,所以RhoG在神经母细胞从SVZ到嗅球首先可能发挥迁移作用,但是这种功能受到miR-124的调控。所以推测miR-124可能通过RhoG调控嗅球颗粒细胞形态合适的发育水平。
[Abstract]:Olfactory system is a part of limbic system, which consists of olfactory epithelium, main olfactory bulb and olfactory cortex.Smell plays an important role in finding food for animals, identifying dangers, and finding spouses and social contacts.Adult subventricular area (SVZ) is one of the neuronal regions in mammalian adult brain.SVZ neuroblastcells migrated to OB and differentiated into granulosa cells (GCs) and peribulbar cells (PGS), and then integrated into the synaptic loop of olfactory bulb.Granulosa cells belong to inhibitory neurons which account for 95% of olfactory bulb intermediate neurons.Their inclusions grow several short basal dendrites in the granular cell layer (GCLs).The apical dendrites straddle the lamina of mitral cells and extend to the outer plexiform layer of EPLN. MicroRNAs belong to Small ncRNs, which are short RNAs with a length of 18-25 nucleotides. MicroRNAs are the major regulatory molecules of post-transcriptional gene expression. MicroRNAs bind to specific mRNA in a sequence complementary manner.The expression of target gene was regulated by degradation of target gene mRNA or inhibition of protein translation.Brain degenerative disease. MiR-124 is a brain-specific miRNAs that account for the 25-48%.miR-124 expression level of miRNA in all brain differentiated and mature neurons in mice.In the central nervous system (CNS) of developing mice, the increasing number of .miR-124 determines the fate of SVZ) neurons in the adult subventricular area.The important function of regulating the regeneration of adult neurons in SVZ. MiR-124 is to regulate the development of neurons and play an important role in neurodegenerative diseases.Small G protein, named for its molecular weight of only 20~30KD, is a class of conjugated proteins located on the medial side of the membrane that bind to the cytosolic domain of the membrane receptor.The membrane receptor ligand binding stimuli are coupled with downstream effector molecules. The RhoGTPases family plays an important role in the morphogenesis of dendritic spine in synaptic plasticity neurons by regulating actin cytoskeleton.The miRNA expression profile of olfactory bulb was revealed by miRNA chip method. It was found that miR-124 was the most expressed miRNA in olfactory bulb.Through target gene prediction and functional network enrichment, we understand that miR-124 plays an important role in regulating cytoskeleton.By constructing miR-124 overexpression vector and inhibiting lentivirus vector, and injecting miR-124 into mouse by stereotactic localization, we demonstrated that miR-124 regulates dendritic morphology and dendritic spine density in the later stage of mouse olfactory bulb granulosa cell development.We found that small G protein RhoG may play an important role in the regulation of cytoskeleton target gene enrichment by miR-124.The constitutive inactivation vector of RhoG and the shRNA lentivirus vector of RhoG were constructed.We observed whether RhoG could promote dendritic development and dendritic spinous density of olfactory bulb granulosa cells by stereotaxic injection of RMS in mice.The results showed that RhoG was activated by downstream Rac1 and phosphorylation of PAK and confilind. The expression pattern of. RhoG was similar to that of miR-124.This seems paradoxical but it can be understood that RhoG may be involved in the regulation of migration, so RhoG may first play a role in migration from SVZ to olfactory bulb in neuroblastocytes, but this function is regulated by miR-124.Therefore, it is speculated that miR-124 may regulate the morphology of olfactory bulb granulosa cells by RhoG.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R339.12

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