MicroRNA regulation of CTGF in hepatic stellate cells

描述（由申请人提供）：在慢性肝损伤期间，肝星状细胞（HSC）产生并沉积过量的原纤维胶原，导致瘢痕形成和肝功能受损。这种纤维化过程是由结缔组织生长因子（CTGF）驱动的。我们广泛的长期目标是抑制CTGF的产生或作用，从而开发出用于人类的有效抗纤维化策略。本应用程序的总体目标是确定microRNA-199a （miR-199a）或microrna -214 （miR-214）在调节CTGF产生中的功能，因为我们发现miR-199a/214是HSC中迄今未被识别的miRs，其功能是CTGF mRNA表达的负调节因子。因此，miR-199a/214在正常肝脏静止HSC中高水平表达，从而抑制CTGF的产生，而在损伤后HSC激活过程中，miR-199a/214的表达被抑制，导致CTGF表达增强。miR -199a/214通过纳米级外泌体从HSC中输出，这是一个重要的新发现，因为它允许miR递送到邻近细胞并调节其中的靶基因。我们的中心假设是，miR-199a/214在HSC中的表达或作用是CTGF mRNA表达的关键决定因素。验证我们假设的目的是：[1]建立调节miR-199a/214在HSC中产生的机制[2]建立miR-199a/214在HSC激活和纤维化中的作用[3]建立外泌体miR-199a/214在细胞间信号传导中的作用这些研究的预期结果将是更完整地了解在HSC激活过程中调节CTGF产生的方式。这项研究的基本原理是，一旦miR-199a/214在CTGF调控中的作用被更好地理解，它们可能被开发为新的抗纤维化药物。新疗法的发展至关重要，因为纤维化病理是目前尚无有效治疗方法的最大疾病群体之一。肝纤维化的医疗和经济负担是巨大的：肝硬化是

DESCRIPTION (provided by applicant): During chronic liver injury, hepatic stellate cells (HSC) produce and deposit excessive quantities of fibrillar collagens leading to scar formation and compromised liver function. This fibrotic process is driven by connective tissue growth factor (CTGF). Our broad long-term objective is to inhibit the production or action or CTGF so that effective anti-fibrotic strategies can be developed for use in humans. The overall objective of this application is to determine the function of microRNA-199a (miR-199a) or mircoRNA-214 (miR-214) in regulating CTGF production in light of our identification of miR-199a/214 as hitherto unrecognized miRs in HSC which function as negative regulators of CTGF mRNA expression. Thus miR-199a/214 are expressed at high levels in quiescent HSC in normal liver thereby inhibiting CTGF production whereas their expression is suppressed during HSC activation after injury leading to enhanced CTGF expression. MiR-199a/214 are exported from HSC via nano-size exosomes, a novel finding that is important since it allows for miR delivery to neighboring cells and regulation of target genes therein. Our central hypothesis is that expression or action of miR-199a/214 in HSC are critical determinants of CTGF mRNA expression. The Aims to test our hypothesis are: [1] Establish mechanisms regulating miR-199a/214 production in HSC [2] Establish the role of miR-199a/214 in HSC activation and fibrosis [3] Establish the role of exosomal miR-199a/214 in intercellular signaling The expected outcome of these studies will be a more complete understanding of the manner in which CTGF production is regulated during HSC activation. The rationale that underlies the proposed research is that once the roles of miR-199a/214 in CTGF regulation are better understood, they may be exploitable as novel anti- fibrotics. Development of new therapies is critical because fibrotic pathology represents one of the largest groups of disorders for which there is no effective therapy. The medical and financial burdens of liver fibrosis are huge: liver cirrhosis is the ninth leading cause of death in the West, affects millions of individuals world- wide, and is a harbinger of hepatic cancer. The proposed studies are responsive to the 2004 trans-NIH "Action Plan for Liver Disease Research and will have a positive impact on improving public health by providing new leads in our understanding of CTGF biology, which is central to the process of liver fibrosis and the development of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Fibrosis of internal organs is a significant contributing factor in about 45% of the deaths in the USA yet there are currently no FDA-approved anti-fibrotic therapeutics. Chronic fibrosis most commonly affects the liver, lung, kidney, heart, pancreas and skin and is the result of diverse causes such as autoimmune, genetic, idiopathic, or toxins (e.g. alcohol in hepatic fibrosis). These studies will determine the role of connective tissue growth factor (CTGF) in controlling the pro-fibrogenic functions of hepatic stellate cells, which are the principal fibrogenic cell type in the liver. This proposal focuses on identifying the mechanisms by which CTGF production is regulated by small naturally occurring RNA molecules called microRNA. The relevance of these studies is that they will identify key steps in the pathways that lead to hepatic fibrosis and will define points of therapeutic intervention that exploit the central role played by connective tissue growth factor (CTGF) in driving fibrotic pathology.