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是必不可少的，因为肝脏是唯一能够 再生。人肝干细胞(HLSCs)具有修复、再生和免疫调节等特性，已被广泛研究。几项使用不同疾病动物模型的研究表明，外源性HLSCs治疗可以缓解包括肝脏疾病在内的急性器官损伤。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。尽管干细胞疗法已被用于肝病的临床前治疗，但对干细胞来源的微囊泡(MVS)及其相关的非编码RNA(NcRNAs)却知之甚少，它们可以介导促进肝病进展和恢复的遗传变化。许多ncRNAs以组织特异性的方式表达，这种方式在人类酒精性肝损伤中发生了异常变化。大多数ncRNAs在肝病中的生物学功能尚不明确。在我们的初步研究中，我们已经发现，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MVs中异常表达，这些基因可以调节对肝损伤的反应以及细胞重塑潜力。基于这些有说服力的数据，我们提出了一个中心假设，即干细胞来源的微泡中的ncRNAs通过诱导肝组织和细胞的生长和重塑而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，并建立了稳定转染或敲除的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来源的微泡，并表征它们在组织修复方面的功能特性。在这一应用中，我们建议系统研究干细胞来源的MVS中具有治疗酒精性肝损伤潜力的干细胞依赖的ncRNAs作为标志物。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，我们将确定在酒精性肝损伤过程中参与存活和细胞衰老的功能性乙醇/内毒素依赖的miRNAs。其次，我们将定义肝细胞中参与组织修复相关细胞功能的功能干性调节的ncRNAs信号。第三，我们将在体内检测茎相关的ncRNAs丰富的微囊对高胆固醇饱和脂肪饮食(HCFD-ALD)/CCL4酒精性肝损伤小鼠的形态和功能恢复的促进作用。将评估抗miRNAs或过表达T-UCRs的干细胞来源的微囊对肝细胞增殖、衰老和纤维化的治疗作用。建议的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，干细胞来源的微泡在酒精性肝损伤过程中对生长和组织修复的调控方面所获得的基础性新知识有望推动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.