Mechanisms of Polyploidy and Aneuploidy in the Liver

描述（由申请人提供）：近2500万美国人受到肝功能障碍的影响，肝脏疾病是美国第十大死亡原因。有一个明确和迫切需要开发新的替代整个器官置换。需要更好地了解肝脏生物学，以改善现有方法并创新治疗肝病的疗法，包括病毒性肝炎和脂肪性肝炎。肝细胞是肝脏中的主要功能性细胞类型，其显示出由普遍的生理多倍性（小鼠中>90%，人类中50%）和非整倍性（小鼠中60%，人类中30-90%）引起的一系列染色体多样性。在真核生物中，细胞通常含有由成对同源染色体组成的二倍体基因组。多倍性是指整组染色体的增加，非整倍性是指单个染色体的增加和损失。肝脏多倍体和非整倍体的作用代表了我们目前对肝脏生物学理解的一个主要空白。我们最近发现，非整倍体增强小鼠肝脏的再生能力。为了应对酪氨酸血症诱导的损伤（通常对肝脏有毒性），我们鉴定了一组对该疾病有抗性的非整倍体肝细胞。这些数据表明，非整倍体肝细胞具有增强的适应和再生能力。我们的中心假设是，非整倍体的功能作为一种适应性机制，对肝损伤的反应。本申请的目的是确定调节肝脏非整倍体/多倍体的机制，并阐明非整倍体如何影响肝功能。为了研究这些问题，我们在具体目标1中提出，确定多倍体肝细胞是否是非整倍体肝脏发育所必需的。实验将表征E2 f7/E2 f8基因敲除小鼠的肝细胞分裂、核型和应激反应，这些小鼠具有正常的肝功能，但缺乏多倍体肝细胞。在具体目标2中，我们将剖析一种新的肝脏多倍性调节因子的作用，该因子最近在我们的实验室中被鉴定为microRNA-122（miR-122）。实验将确定miR-122如何在整个生命过程中改变倍性和非整倍性。我们还将确定miR-122调节肝倍性的细胞和分子机制。最后，在具体目标3中，我们将确定随机核型（在非整倍体肝细胞中）如何影响肝脏功能。我们将利用一种新的异种移植模型来研究再生人肝细胞的克隆结节。实验将测量非整倍体并确定这些结节中的基因表达谱。总之，这些研究将确定非整倍体影响肝脏修复/再生的程度以及控制这一过程的分子机制。了解非整倍体肝细胞如何产生和功能将为肝脏稳态，疾病和治疗提供新的和关键的见解。

DESCRIPTION (provided by applicant): Nearly 25 million Americans are affected by liver dysfunction, and liver diseases are the 10th leading cause of death in the US. There is a clear and urgent need for developing new alternatives to whole organ replacement. A better understanding of liver biology is required to improve existing approaches and to innovate therapies for the treatment of liver diseases, including viral hepatitis and steatohepatitis. Hepatocytes, the primary functional cell type in the liver, display a range of chromosomal diversity resulting from prevalent physiological polyploidy (>90% in mice and 50% in humans) and aneuploidy (60% in mice and 30-90% in humans). In eukaryotic organisms, cells usually contain a diploid genome comprised of pairs of homologous chromosomes. Polyploidy refers to gains in entire sets of chromosomes, and aneuploidy refers to gains and losses of individual chromosomes. The roles of hepatic polyploidy and aneuploidy represent a major gap in our current understanding of liver biology. We recently found that aneuploidy enhances the regenerative capacity of the mouse liver. In response to Tyrosinemia-induced injury, that is normally toxic to the liver, we identified a subset of aneuploid hepatocytes that was resistant to the disease. The data suggest that aneuploid hepatocytes are endowed with enhanced capacity for adaptation and regeneration. Our central hypothesis is that aneuploidy functions as an adaptive mechanism in response to hepatic injury. The goals of this application are to identify mechanisms regulating hepatic aneuploidy/polyploidy and to unravel how aneuploidy affects liver function. To investigate these questions, we propose in Specific Aim 1 to determine whether polyploid hepatocytes are necessary for development of aneuploid livers. Experiments will characterize hepatic cell divisions, karyotypes and stress response in E2f7/E2f8 knockout mice, which have normal liver function but are depleted of polyploid hepatocytes. In Specific Aim 2, we will dissect the role of a novel regulator of hepatic polyploidy, recently identified in ur laboratory, microRNA-122 (miR-122). Experiments will determine how miR-122 alters ploidy and aneuploidy throughout life. We will also identify cellular and molecular mechanisms by which miR-122 regulates hepatic ploidy. Finally, in Specific Aim 3, we will determine how random karyotypes (in aneuploid hepatocytes) affect function in the liver. We will utilize a novel xenotransplantation model to examine clonal nodules of regenerating human hepatocytes. Experiments will measure aneuploidy and determine gene expression profiles in these nodules. Together, these studies will define the extent to which aneuploidy affects liver repair/regeneration as well as the molecular mechanisms that control this process. Understanding how aneuploid hepatocytes arise and function will provide new and crucial insights into liver homeostasis, diseases and treatments.