Long noncoding RNAs regulating liver fibrosis

项目摘要 长非编码(LNC)RNA是我们必须探索的一个新的前沿，以充分了解肝纤维化。这节课 与信使(M)RNA具有相同的功能，但不是编码蛋白质，而是lncRNAs 来调节细胞的功能。到目前为止，我们只了解到一小部分lncRNAs在 肝星状细胞(HSCs)，是导致纤维化瘢痕产生的主要细胞类型。接下来的 在我们对造血干细胞中lncRNA功能的认识上存在这种根本的鸿沟，将阻碍我们发展的能力。 治疗肝纤维化的新方法。这项工作的长期目标是了解一个新的lncRNA是如何 我们已经确定(Tilac，转化生长因子-β诱导的lncRNA激活胶原)调节胶原(COL1A1)的产生 在肝干细胞中控制肝纤维化的进展，并利用这一见解开发治疗患者的新方法 患有慢性肝病。我们通过对人HSCs中的Tilac进行RNA测序分析来鉴定Tilac。我们发现 它是由纤维化信号转化生长因子-β诱导的，仅限于HSC表达，在人肝纤维化中被激活。 丁香酸的消耗导致I型胶原表达减少，I型胶原是纤维性瘢痕的主要成分。我们 已经通过Tilac的保守基因组位置鉴定了Tilac的小鼠同源基因，并发现这个lncRNA是 诱导小鼠肝星状细胞发生体内纤维化。丁香酸在小鼠造血干细胞中的耗竭也是 与I型胶原表达降低有关。我们的总体目标是确定Tilac如何监管 肝纤维化。核心假设是Tilac通过调节COL1A1来控制纤维化的进展 在人和小鼠的HSCs中表达，并可靶向抑制纤维化。这项提议的理由是 了解Tilac的功能并开发一个模型来研究Tilac在体内的活动将提供关键 洞察incRNAs如何调节肝纤维化。这项提议还将定义一种独特的治疗目标 在HSCs中在肝脏中表达，并且可以在不影响肝脏其他细胞类型的情况下被耗尽。中环 假说将通过追求两个具体目标来检验：(1)确定Tilac如何调节 COL1A1和(2)确定了Tilac在体内肝纤维化中的作用。在第一个目标中，功能丧失 RNA荧光原位杂交和lncRNA沉淀相结合的分析与挽救实验 将决定丁香酸如何控制I型胶原的表达和纤维化。在第二个目标中，我们将使用丁香- 报告和Tilac缺陷小鼠在体内定义表达Tilac的细胞类型并确定Tilac如何 调节肝纤维化的功能。拟议的工作意义重大，因为通过 哪些lncRNA调节肝纤维化将激发变革性的策略，通过调节 InncRNA。它还将建立一条在小鼠身上识别和研究与患者最相关的lncRNAs的途径 慢性肝病。本工作的创新之处在于聚焦于lncRNAs对肝纤维化发病机制的认识。 以及它如何应用基因组编辑方法来干扰和跟踪lncRNA的表达。

Project Summary Long noncoding (lnc) RNAs are a new frontier that we must explore to fully understand liver fibrosis. This class of noncoding RNAs has the same features as messenger (m) RNAs, but instead of encoding protein, lncRNAs operate to regulate cellular functions. To date, we understand the activity of only a small number of lncRNAs in hepatic stellate cells (HSCs), the primary cell type responsible for production of the fibrotic scar. The continued existence of this fundamental gap in our knowledge of lncRNA function in HSCs will impede our ability to develop new approaches to treat liver fibrosis. The long-term goal of this work is to understand how a new lncRNA that we have identified (TILAC, TGF-b-induced lncRNA Activating Collagen) regulates collagen (COL1A1) production in HSCs to control progression of liver fibrosis and use this insight to develop new approaches to treat patients with chronic liver disease. We identified TILAC through RNA-sequencing analysis in human HSCs. We find that it is induced by the fibrotic signal TGF-b, restricted in expression to HSCs and activated in human liver fibrosis. Depletion of TILAC leads to reduced expression of type I collagen, a primary component of the fibrotic scar. We have identified the mouse ortholog of TILAC by its conserved genomic location and find that this lncRNA is induced in murine HSCs with in vivo development of fibrosis. Depletion of TILAC in murine HSCs is also associated with decreased type I collagen expression. Our overall objective is to determine how TILAC regulates liver fibrosis. Out central hypothesis is that TILAC controls progression of fibrosis through regulation of COL1A1 expression in human and murine HSCs and can be targeted to inhibit fibrosis. The rationale for this proposal is that understanding how TILAC functions and developing a model to study TILAC activity in vivo will provide key insight into how lncRNAs regulate liver fibrosis. This proposal will also define a therapeutic target that is uniquely expressed in the liver in HSCs, and could be depleted without affecting other cell types in the liver. The central hypothesis will be tested by pursuing two specific aims: (1) Determine how TILAC regulates expression of COL1A1 in human HSCs and (2) Define the role of TILAC in liver fibrosis in vivo. In the first aim, loss-of-function analysis and rescue experiments coupled with RNA fluorescent in situ hybridization and lncRNA precipitation will determine how TILAC controls type I collagen expression and fibrosis. In the second aim, we will use TILAC- reporter and TILAC-deficient mice to define the cell types that express TILAC in vivo and determine how TILAC functions to regulate liver fibrosis. The proposed work is significant because understanding the mechanism by which lncRNAs regulate liver fibrosis will inspire transformative strategies to inhibit fibrosis through regulation of lncRNAs. It will also establish a path to identify and study lncRNAs in mice that are most relevant to patients with chronic liver disease. This work is innovative in the focus on lncRNAs to understand mechanism of liver fibrosis and how it applies genome editing approaches to disrupt and track lncRNA expression.