Long noncoding RNAs regulating liver fibrosis

项目摘要 长链非编码RNA（lnc）是一个新的前沿，我们必须探索，以充分了解肝纤维化。这个类 的非编码RNA具有与信使（m）RNA相同的特征，但不是编码蛋白质，lncRNA 调节细胞功能。到目前为止，我们只了解了一小部分lncRNA在细胞中的活性。 肝星状细胞（HSC），负责产生纤维化瘢痕的主要细胞类型。继续 在我们对HSC中lncRNA功能的认识中存在这种根本性的差距将阻碍我们发展HSC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种新的lncRNA， 我们已经鉴定出（TILAC，TGF-β诱导的lncRNA激活胶原蛋白）调节胶原蛋白（COL 1A 1）的产生 在HSC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人HSC中的RNA测序分析鉴定了TILAC。我们发现 它由纤维化信号TGF-β诱导，限制在HSC中表达并在人肝纤维化中活化。 TILAC的消耗导致I型胶原蛋白（纤维化瘢痕的主要成分）的表达减少。我们 通过其保守的基因组位置鉴定了TILAC的小鼠直系同源物，并发现这种lncRNA是 在鼠HSC中诱导，体内发生纤维化。鼠HSC中TILAC的耗尽也是 与I型胶原蛋白表达减少有关。我们的总体目标是确定TILAC如何调节 肝纤维化我们的中心假设是TILAC通过调节COL 1A 1来控制纤维化的进展 在人和鼠HSC中表达，并且可以靶向抑制纤维化。这项建议的理由是 了解TILAC的功能并开发一个模型来研究体内TILAC的活性将为我们提供关键的 lncRNA如何调节肝纤维化的研究进展该提案还将确定一个治疗靶点， 在HSC的肝脏中表达，并且可以在不影响肝脏中其他细胞类型的情况下被耗尽。中央 将通过追求两个具体目标来检验假设：（1）确定TILAC如何调节 COL 1A 1在人HSC中的作用和（2）确定TILAC在体内肝纤维化中的作用。在第一个目标中，功能丧失 结合RNA荧光原位杂交和lncRNA沉淀的分析和拯救实验 将决定TILAC如何控制I型胶原表达和纤维化。在第二个目标中，我们将使用TILAC- 报告基因和TILAC缺陷型小鼠，以确定体内表达TILAC的细胞类型，并确定TILAC 调节肝纤维化的功能。所提出的工作是有意义的，因为了解的机制， 哪些lncRNA调节肝纤维化将激发通过调节 lncRNA。它还将建立一种途径，以识别和研究小鼠中与患者最相关的lncRNA。 慢性肝病这项工作是创新的重点lncRNAs了解肝纤维化的机制 以及它如何应用基因组编辑方法来破坏和跟踪lncRNA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.