Investigating the Role of IGF2 mRNA-binding protein 1 (IMP1) In Regulating Reserve Intestinal Stem Cell Survival In Response to DNA Damage

研究 IGF2 mRNA 结合蛋白 1 (IMP1) 在调节储备肠干细胞响应 DNA 损伤的存活中的作用

• 批准号: 10326363
• 负责人: Louis Parham
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10326363
• 关键词: Ablation; Adult; Adverse effects; Affect; Alleles; Antioxidants; Apoptosis; Architecture; Autophagocytosis; Binding Proteins; CASP3 gene; Cell Count; Cell Cycle; Cell Death; Cell Maintenance; Cells; Cessation of life; Cleaved cell; Cysteine; DNA Damage; Data; Development; Embryonic Development; Epithelial; Epithelial Cells; Exhibits; Flow Cytometry; Foundations; Frequencies; Gene Expression; Genes; Homeostasis; Hour; IGF2 gene; Immunohistochemistry; Impairment; In Situ Nick-End Labeling; Injury; Intestines; Knock-out; Knockout Mice; Knowledge; Label; Malignant Neoplasms; Measures; Mediating; Membrane Potentials; Messenger RNA; Mitochondria; Mus; Natural regeneration; Organ; Pathologic; Pathway interactions; Perinatal mortality demographics; Phenocopy; Play; Polyadenylation; Population; RNA Splicing; RNA-Binding Proteins; Radiation; Radiation Dose Unit; Radiation therapy; Reactive Oxygen Species; Recovery; Regenerative response; Regulator Genes; Reporter; Role; Syndrome; Tamoxifen; Testing; Therapeutic; Transcript; Vesicle; Whole-Body Irradiation; base; cell behavior; cell regeneration; cell type; epithelium regeneration; experimental study; gastrointestinal; intestinal crypt; intestinal epithelium; intestinal homeostasis; irradiation; loss of function; mitotracker green FM; novel therapeutics; radiation resistance; radioresistant; regenerative; resilience; response; self renewing cell; stem cell model; stem cell survival; stem cells; tissue injury; tissue regeneration; transcriptome sequencing

The intestinal epithelium undergoes complete turnover in 3-5 days. This is achieved through the presence of active intestinal stem cells (a-ISCs) that are self-renewing and can give rise to all differentiated epithelial cell types. Following irradiation, which ablates cycling cells in the intestine, cells that survive irradiation referred to as reserve intestinal stem cells (r-ISCs) are able to re-enter the cell cycle and contribute to tissue regeneration. Although mechanisms regulating the dormancy of these cells are beginning to be elucidated, their radioresistance and subsequent re-activation is still unclear, representing a gap in our broader knowledge of intestinal epithelial regeneration. RNA-binding proteins (RBP) are regulators of gene expression that facilitate cellular adaptation during homeostasis or pathological states. Deletion of the RBP IGF2 mRNA-binding protein 1 (IMP1) in the intestinal epithelium is associated with enhanced regenerative response following irradiation, a result that is phenocopied in mice with Imp1 deleted specifically in r-ISCs, highlighting a putative role for Imp1 in r-ISCs. Imp1 loss in the intestinal epithelium leads to an increase in autophagic vesicle content at homeostasis, suggesting that Imp1 may function as a negative regulator of the autophagy pathway. Combined deletion of Imp1 and Atg7 in the intestinal epithelium abrogates the enhanced regeneration observed with Imp1 deletion, suggesting that Imp1’s regulatory roles are dependent on the autophagy pathway. Studies show that mice lacking the autophagy gene Atg5 in intestinal epithelial cells exhibit a marked decrease in a-ISCs and impaired regeneration following irradiation. This is attributed to accumulation of dysfunctional mytochondria and a subsequent increase in reactive oxygen species levels (ROS). The role played by autophagy in radioresistant r- ISCs during homeostasis and regeneration remains unknown. I hypothesize that Imp1 modulates r-ISC survival in response to DNA-damage by enhancing autophagy mediated clearing of reactive oxygen species. In Aim 1, we will evaluate r-ISC frequency at homeostasis as well as cell death and DNA damage post- irradiation with or without Imp1 deletion. We will use a reporter mouse that labels r-ISCs and progeny with RFP following tamoxifen administration for lineage-tracing. We will use 12Gy whole-body irradiation and evaluate RFP+ cells using flow cytometry and immunohistochemistry. For Aim 2 we will use r-ISC specific Atg7 knockout as well as Atg7 and Imp1 double knockout mice to determine whether enhanced regeneration observed in the absence of Imp1 is dependent on autophagy. Regenerative microcolony frequency will be measured via immunohistochemistry. Furthermore, we will evaluate how Imp1 loss affects mitochondrial content using Mitotracker Green FM, transmembrane potential using rhodamine123, and ROS using CellROX via flow cytometry. Lastly, we will evaluate how ROS modulation affects regenerative microcolony formation with or without the antioxidant N-acetyl-L-cysteine. These studies will uncover roles played by RBPs in regulating stem cell activity and will provide a mechanistic understanding of stem cell survival following DNA damage.

肠上皮在3-5天内完成翻转。这是通过以下方式实现的 活跃的肠道干细胞(a-ISCs)能够自我更新，并能分化为所有分化的上皮细胞 类型。在照射后，肠道中的循环细胞被破坏，在照射后存活的细胞被称为 作为储备的肠道干细胞(r-ISCs)能够重新进入细胞周期，促进组织再生。 尽管调控这些细胞休眠的机制开始被阐明，但它们的 辐射抗性和随后的重新激活仍然不清楚，这代表着我们对 肠上皮再生。RNA结合蛋白(RBP)是基因表达的调节者，有助于 细胞在动态平衡或病理状态下的适应。RBP IGF2 mRNA结合蛋白的缺失 肠上皮细胞中的1(IMP1)与辐射后增强的再生反应有关，a 结果显示，在r-ISCs中Imp1特异性缺失的小鼠中，突出了Imp1在 R-ISCs。肠上皮中Imp1的丢失导致自噬小泡含量在稳态时增加， 提示Imp1可能是自噬途径的负调控因子。Imp1的组合删除 肠上皮细胞中的ATG7消除了Imp1缺失所观察到的增强的再生， 提示Imp1‘S的调控作用依赖于自噬途径。研究表明，老鼠 缺乏自噬基因ATG5的肠上皮细胞表现出a-ISCs显著减少和受损 辐射后的再生。这归因于功能障碍的肌软骨症的积累和 随后活性氧物种水平(ROS)增加。自噬在辐射抗性中的作用 ISCS在动态平衡和再生过程中的作用尚不清楚。我假设Imp1调节r-ISC 通过增强自噬介导的活性氧清除来应对DNA损伤的存活 物种。在目标1中，我们将评估r-ISC在动态平衡以及细胞死亡和DNA损伤后的频率。 辐射后Imp1缺失或不缺失。我们将使用一个报告鼠标，用RFP标记r-ISCs和后代 使用他莫昔芬进行血统追踪。我们将使用12Gy全身照射，并评估 用流式细胞仪和免疫组织化学方法检测RFP+细胞。对于目标2，我们将使用r-ISC特定的ATG7基因敲除 以及ATG7和Imp1双基因敲除小鼠，以确定在 Imp1的缺失依赖于自噬。再生微菌落频率将通过以下方式测量 免疫组织化学。此外，我们将评估Imp1缺失如何影响线粒体含量 MitoTracker Green FM，使用罗丹明123的跨膜电位，以及使用CellROX Via Flow的ROS 细胞学。最后，我们将评估ROS调节如何影响再生微集落形成或 不含抗氧化剂N-乙酰-L-半胱氨酸。这些研究将揭示限制性商业惯例在调节茎中所起的作用 并将提供对干细胞在DNA损伤后存活的机制的理解。