Long non-coding RNAs in Islet Cell Biology

目前正在进行大量的研究工作，以了解和预防糖尿病。为了实现这一目标，对小鼠的研究极大地促进了我们对保守的信号通路和调节因子的理解，这些信号通路和调节因子是功能β细胞发展和维持所需的。然而，从目前与预测和治疗糖尿病以及产生替代内分泌细胞来源相关的挑战中可以明显看出，我们仍然缺少产生、成熟和保存全功能β细胞所需的关键分子成分。近年来，基因组生物学的研究进展表明，哺乳动物基因组的很大一部分是转录的，其中包括大量的长非编码RNA(LncRNAs)，其中许多是在小鼠和人类之间保守的。LncRNAs是一组不同的转录本，长度超过200个核苷酸，类似于mRNAs，但不编码蛋白质。有新的证据表明，lncRNA可能与β细胞功能和糖尿病有关。为了支持这一数据，已确定大多数与糖尿病相关的SNP映射到基因组的非编码区，其中许多特定位于LncRNAs中。因此，保守的胰岛特异性LncRNA的鉴定和鉴定将大大加深我们对控制β细胞发育和功能的调控网络的理解，以及这些机制的破坏如何导致糖尿病。在这项研究中，我们使用比较基因表达分析和新的计算算法来识别和排序在人和小鼠胰腺中表达的潜在相关的lncRNAs。我们已经选择了其中的两个LncRNA进行进一步的功能分析，基于一套定义的标准，包括它们的表达阶段和水平，胰岛特异性，小鼠和人类之间的同时性和保守性，与胰腺相关的编码基因的接近，以及与糖尿病相关的SNPs的接近。这项提议的总体目标是揭示选定的lncRNA在胰岛和β细胞生物学中的功能作用。我们假设，长的非编码RNA(LncRNAs)是胰岛转录调控途径中缺失的组成部分，在β细胞的发育和功能中发挥重要作用。为了验证这一假说，我们将鉴定βlinc1的功能和分子活性，它是一种在小鼠和人类胰岛中表达的新的lncRNA。我们已经产生了βlinc1缺失的小鼠，并将鉴定βlinc1的KO表型，并确定βlinc1的分子活性。我们还鉴定了PAUPAR是一种保守的LncRNA，在表达胰高血糖素的成人α细胞中高度表达。此外，Paupar还调节Pax6的表达和一些Pax6独立和依赖的转录活动。我们通过在Paupar基因座上插入一个H_2B-GFP盒产生了Paupar的零等位基因。我们拟对Paupar在胰岛中的表达、功能作用和分子机制进行研究。

A large number of research efforts are currently underway to understand and prevent diabetes. Towards this goal, studies in mice have significantly advanced our understanding of the conserved signaling pathways and regulatory factors required for the development and maintenance of functional β cells. However, it is evident from the current challenges associated with predicting and treating diabetes, and generating alternative sources of endocrine cells, that we are still missing key molecular components that are required to generate, mature and preserve fully functional β cells. Recently, advances in genome biology have revealed that a large part of the mammalian genome is transcribed and includes a large number of long non-coding RNAs (lncRNAs), many which are conserved between mouse and human. LncRNAs are a diverse group of transcripts longer than 200 nucleotides that resemble mRNAs, but do not encode proteins. There is emerging evidence to suggest lncRNAs may be involved in β cell function and diabetes. In support of this data, it has been determined that the majority of diabetes-associated SNPs map to non-coding regions of the genome, many that are specifically located in lncRNAs. Therefore, the identification and characterization of conserved islet-specific lncRNAs will significantly further our understanding of the regulatory networks that control β cell development and function, and how disruption of these mechanisms may lead to diabetes. For this study, we have used comparative gene expression analyses and novel computational algorithms to identify and rank potentially relevant lncRNAs expressed in the human and mouse pancreas. We have selected two of these lncRNAs for further functional analysis based on a defined set of criteria, including their stage and level of expression, islet specificity, synteny and conservation between mouse and human, proximity to pancreas-related coding genes, and proximity to SNPs associated with diabetes. The overall goal of this proposal is to reveal the functional roles of selected lncRNAs in islet and β cell biology. We hypothesize that long non-coding RNAs (lncRNAs) represent a missing component of the islet transcriptional regulatory pathways and play essential roles in β cell development and function. To test this hypothesis, we will characterize the function and molecular activity of βlinc1, a novel lncRNA expressed in mouse and human islets. We have generated mice that are deleted for βlinc1 and will characterize the βlinc1 KO phenotype and determine the molecular activity of βlinc1. We have also identified Paupar as a conserved lncRNA that is highly expressed in adult glucagon-expressing α cells. In addition, Paupar regulates Pax6 expression and a number of Pax6-independent and dependent transcriptional activities. We have generated a null allele of Paupar by inserting an H2B-GFP cassette into the Paupar locus. We propose to characterize the expression, functional role and molecular mechanism of Paupar in the pancreatic islet.