Role of the Snail1-Twist-p21 axis on cell cycle arrest and renal fibrosis development

慢性肾脏疾病(CKD)，如肾脏纤维化，是全球健康挑战。仅在美国就有CKD费用 2013年，为65岁及以上的慢性肾脏病患者提供的医疗保险估计为500亿美元。最新研究 显示在AKI后，肾上皮细胞经历了部分上皮向间充质转化(PEMT)和 通过Snail1-Twist1-p21轴的G2/M细胞周期停滞；这些细胞分泌促纤维化因子，并有助于 纤维化进展。因此这三种因子成为治疗纤维化的有希望的潜在药物靶点， 但进一步的发展需要解决几个悬而未决的问题。时间序列和 PEMT与细胞周期停滞之间的因果关系存在争议，Snail1，Twist1， 而p21在调节PEMT和细胞周期停滞方面的作用尚不清楚。解决这些问题需要量化 系统生物学的方法超越了该领域传统使用的细胞生物学方法。最近几年我的实验室 在基于深度学习的活细胞图像自动分析方面取得了进展，基于CRISPR 基因编辑、数学建模和其他定量生物学工具。这些技术 事态的发展使我们能够解决与肾脏纤维化有关的上述挑战性问题。 根据现有的研究和我们的初步结果，我们假设存在一个时间顺序 三个因素，其中p21启动了G2/M期停滞，随后Snail1；Snail1的上调加强了这一点 也被激活，并且Twist1进一步维护PEMT计划；由于它们的角色在时间上变化， 针对这些因素的有效性取决于治疗的时机。我们将用以下方法检验该假设 利用已建立的细胞系和原代肾上皮细胞进行定量成像研究 竞争模型的分析。在目标1中，我们将进行多色流式细胞术研究和延时 研究细胞在刺激下的细胞周期、EMT和其他细胞命运的成像研究。这两种类型 将提供关于PEMT和细胞周期是否紧密耦合的补充信息，并将 绘制出各种细胞命运变化事件的时间序列以及与表达水平的相关性 三个因素中的一个。在目标2中，我们将通过荧光蛋白来监测这些因子的时间分布 在单个单元格中进行标记，并使用数据来评估一组模型以识别一个或一组最小 监管EMT和G2/M停滞的网络。然后，我们将通过以下方式进一步研究个别因素的作用 模型分析和一系列抑制实验。 拟议研究的成功将提供对细胞调控网络的机械性理解 肾上皮细胞周期停滞与EMT拟议中的研究是我们对一个新兴领域的起点 肾脏纤维化的定量系统生物学研究。我们预计，引入量化方法将 大大加快未来治疗战略的发展，以应对日益严重的全球健康挑战 由于纤维化的进展，目前缺乏有效的治疗。

Chronic kidney diseases (CKD) such as kidney fibrosis are global health challenges. In the US alone CKD cost Medicare an estimated 50 billion dollars for patients with CKD age 65 and older in 2013. Recent studies revealed that after AKI renal epithelial cells undergo a partial epithelial-to-mesenchymal transition (pEMT) and G2/M cell cycle arrest through a Snail1-Twist1-p21 axis; these cells secrete profibrotic factors and contribute to fibrosis progression. Therefore these three factors become promising potential drug targets for treating fibrosis, but further development requires addressing several outstanding open questions. The temporal sequence and causal relation between pEMT and cell cycle arrest is controversial, and the respective roles of Snail1, Twist1, and p21 on regulating pEMT and cell cycle arrest is unclear. Addressing these questions requires quantitative systems biology approaches beyond cell biology methods traditionally used in the field. In recent years my lab has made progression on deep learning based image automated analysis for live cell images, CRISPR-based gene editing, and mathematical modeling and other quantitative biology tools. These technological developments position us to tackle the above-mentioned challenging questions related to kidney fibrosis. Based on existing studies and our preliminary results, we hypothesize that there is a temporal order of the three factors, with p21 initializing G2/M arrest, which is reinforced by subsequent upregulation of Snail1; Snail1 also activates, and Twist1 further maintains the pEMT program; due to their temporally varying roles, effectiveness of targeting these factors depends on the timing of treatment. We will test the hypothesis with quantitative imaging studies using established cell lines and primary renal epithelial cells and mathematical analysis of competing models. In Aim 1, we will perform multi-color flow cytometry studies and time-lapse imaging studies on progression of cell cycle, EMT, and other cell fates of cells under stimulation. The two types of studies will provide complementary information on whether pEMT and cell cycle are tightly coupled, and will map out the temporal sequence of events of various cell fate change as well as correlation to expression levels of the three factors. In Aim 2, we will monitor the temporal profiles of these factors through fluorescence protein tagging in single cells, and use the data to evaluate an ensemble of models to identify one or a set of minimal network regulating EMT and G2/M arrest. We will then further examine the roles of individual factors through model analysis and a series of inhibition experiments. Success of the proposed research will provide mechanistic understanding of the regulatory network of cell cycle arrest and EMT in renal epithelial cells. The proposed research is our starting point for an emerging field of quantitative systems biology on kidney fibrosis. We expect that introducing quantitative approaches will greatly accelerate future development of treatment strategies on the increasing global health challenge imposed by progression of fibrosis, which currently lacks effective treatment.