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天内进行完全营业额。 活性肠干细胞（A-ISC）是自我更新，可以给予所有不同的上皮细胞细胞细胞细胞细胞 辐射后的类型 由于储备肠道干细胞（R-ISC）能够重新进入周期并有助于组织再生。 尽管调节这些名人的休眠的机制开始被阐明，但 放射线和随后的重新激活仍是叔叔 肠上皮再生。 稳态或病理状态期间的细胞适应。 肠上皮中的1（IMP1）在照射后与再生反应增强，A 由ITH IMP1专门在R-ISC中删除的小鼠中进行表演的结果，突出了IMP1在 r-siscs。 提示IMP1可能是自噬途径的负调节剂。 肠上皮中的ATG7消除了用IMP1划痕观察到的增强的再生， 提示IMP1的调节作用取决于自噬途径。 在肠上皮中缺少自噬基因ATG5 A-ISCS明显下降和受损 辐照后的再生。 随后的活性氧水平（ROS）的增加。 稳态期间的ISC仍然未知。 通过增强自噬介导的清除氧来应对DNA破坏的生存 在AIM 1中，我们将评估稳态的R-ISC频率以及细胞死亡和损害 有或没有IMP1的辐照。 他莫昔芬进行谱系追踪。 RFP+细胞使用流式细胞仪和免疫组织化学。 以及ATG7和IMP1双基因敲除小鼠，以确定您是否在TEE中观察到了增强的恢复 IMP1的缺乏取决于自噬。 免疫组织化学。 mitracker绿色FM，使用若丹明123的跨膜电势，以及使用Cellrox通过流量的ROS 最后，我们将评估ROS调制如何影响使用或或或或或或或或或或或或或或或以或或或或或或或或或或或或 如果没有抗氧化剂N-乙酰L-半胱氨酸。 细胞活性，并将提供对DNA损伤后干细胞存活的机械理解。