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.
The proposed research is relevant to public health because understanding the function of long noncoding RNAs that regulate production of collagen in hepatic stellate cells will improve our ability to inhibit progression of liver fibrosis. The identification of long noncoding RNAs that are conserved between humans and mice will also lead to the development of in vivo models to study lncRNAs relevant to human disease. Thus, the proposed project is relevant to the NIH?s mission to seek and apply knowledge to lengthen life and reduce illness.
