Functional characters of non-coding RNAs in alcoholic liver injury

描述（由申请人提供）： 人类肝脏的功能对生命至关重要，因为肝脏是唯一能够 再生人类肝脏干细胞（HLSC）因其修复、再生和免疫调节特性而被广泛研究。使用不同疾病动物模型的几项研究表明，用外源性HLSC治疗可改善急性器官损伤，包括肝脏疾病。其机制可能涉及旁分泌因子促进存活的固有上皮细胞的增殖。虽然干细胞疗法已经在临床前用于治疗肝脏疾病，但对干细胞衍生的微泡（MV）及其相关的非编码RNA（ncRNA）知之甚少，其可以介导促进肝脏疾病进展和恢复的遗传变化。许多ncRNA以组织特异性方式表达，在人类酒精性肝损伤中发生异常改变。大多数ncRNA在肝脏疾病中的生物学功能尚不明确。在我们的初步研究中，我们已经表明，选定的ncRNA基因在酒精性肝损伤后发生改变，并在人肝干细胞及其衍生的MV中异常表达，可以调节对肝损伤的反应以及细胞重塑潜力。基于这些令人信服的数据，我们提出了一个中心假设，即干细胞衍生的微泡中的ncRNA通过诱导肝组织和细胞的生长和重塑有助于酒精性肝损伤的恢复。为了验证这一假设，我们建立了ncRNA基因操作，功能研究和相互作用分析的技术，并产生稳定转染或敲低细胞系以及酒精性肝损伤的动物模型。我们的长期目标是鉴定和分离干细胞衍生的微泡，并表征其组织修复的功能特性。在本申请中，我们提出了干细胞依赖性ncRNA作为干细胞衍生的MV中具有治疗酒精性肝损伤潜力的标志物的系统研究。我们将通过关注三个具体目标来解决我们的中心假设。首先，我们将确定功能性乙醇/LPS依赖性miRNA参与酒精性肝损伤期间的存活和细胞衰老。第二，我们将定义在肝细胞中参与组织修复相关细胞功能的功能性干性调节的ncRNA信号传导。第三，我们将在体内确定干性相关的ncRNA富集的微泡对加速具有高胆固醇和饱和脂肪饮食（HCFD-ALD）/CCl 4的ALD/ALI小鼠中酒精性肝损伤的形态和功能恢复的作用。将评估源自具有抗miRNA或过表达T-UCR的干细胞的微泡对肝细胞增殖、衰老和纤维化的治疗作用。这些研究结果可能会为人类酒精性肝损伤提供合理的治疗策略。同时，获得的关于通过干细胞衍生的微泡在酒精性肝损伤期间调节生长和组织修复的基础新知识有望推进干细胞生物学的一般领域。

DESCRIPTION (provided by applicant): The functions of the human liver are essential to life since the liver is the only organ capable of regeneration. Human liver stem cells (HLSCs) have been extensively studied for their reparative, regenerative and immunomodulatory properties. Several studies, using different animal models of diseases, showed that treatment with exogenous HLSCs ameliorates acute organ injury including hepatic disorders. The mechanisms may involve paracrine factors promoting proliferation of surviving intrinsic epithelial cells. While stem cell therapies have bee in pre-clinical use for the treatment of liver diseases, very little is known about the stem cell derived microvesicles (MVs) and their related non-coding RNAs (ncRNAs), which can mediate genetic changes that promote the progression and recovery of liver disorders. Many ncRNAs are expressed in a tissue-specific manner that is aberrantly altered in human alcoholic liver injuries. The biological function of the majority of ncRNAs in liver diseases is undefined. In our preliminary studies, we have shown that selected ncRNA genes are altered after alcoholic liver injuries, and aberrantly expressed in human liver stem cells and their derived MVs that can modulate the response to liver injury as well as cell remodeling potentials. Based on these compelling data, we propose the central hypothesis that ncRNAs in stem cell derived microvesicles contribute to the recovery of alcoholic liver injury through induction of growth and remodeling of hepatic tissues and cells. To test this hypothesis, we have established techniques for ncRNA gene manipulation, functional investigation and interaction analysis, and generated stably transfected or knockdown cell lines as well as animal models of alcoholic liver injury. Our long-term objective is to identify and isolate stem cell derived microvesicles and characterize their functional properties of tissue repair. In this application, we propose the systematic investigation of stemness dependent ncRNAs as markers in stem cell derived MVs with the therapeutic potentials for alcoholic liver injury. We will address our central hypothesis by focusing on three specific aims. First, we will identify functional ethanol/LPS dependent miRNAs involved in survival and cellular senescence during alcoholic liver injury. Second, we will define the functional stemness regulated ncRNAs signaling involved in tissue repair-related cellular functions in hepatic cells. Third, we will determine the effects of stemness related ncRNAs enriched microvesicles on accelerating the morphologic and functional recovery of alcoholic liver injury in ALD/ALI mice with high cholesterol and saturated fat diet (HCFD- ALD)/CCl4 in vivo. Therapeutic effects of microvesicles derived from stem cells with anti-miRNAs or over- expression of T-UCRs on hepatic cell proliferation, senescence and fibrosis will be evaluated. The results of proposed studies may lead to rational therapeutic strategies for human alcoholic liver injury. Meanwhile, the acquired fundamental new knowledge about regulation of growth and tissue repair during alcoholic liver damage by stem cell derived microvesicles is expected to advance the general field of stem cell biology.