Impact of in utero diabetes exposure on miRNA: effects on cellular metabolism

子宫内糖尿病暴露对 miRNA 的影响：对细胞代谢的影响

• 批准号: 10217867
• 负责人: Jeanie Beatrice Tryggestad
• 金额: $ 9.45万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217867
• 关键词: Adipocytes; Adipose tissue; Affect; Animal Model; Binding; Biological; Biological Assay; Blood Circulation; Cardiometabolic Disease; Cell Cycle Arrest; Cell Lineage; Cell physiology; Cells; Child; Data; Development; Diabetes Mellitus; Disease; Elements; Endothelial Cells; Epigenetic Process; Event; Exposure to; Fetal Development; Fetal Tissues; Flow Cytometry; Future; Glucose; Goals; Health; High-Throughput Nucleotide Sequencing; Human; IRS1 gene; Immunoprecipitation; Impairment; In Vitro; Infant; Insulin Resistance; Insulin Signaling Pathway; Isotopes; Longevity; Measures; Mediator of activation protein; Mesenchymal Stem Cells; Messenger RNA; Metabolic; Metabolic Diseases; Metabolic Pathway; Metabolism; Methods; MicroRNAs; Newborn Infant; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Pathway interactions; Perinatal; Placenta; Pregnancy; Prevention; Proteins; RNA; Repression; Research; Risk; Role; Testing; Tissues; Transfection; Translations; Umbilical vein; Western Blotting; Work; Youth; adipocyte differentiation; base; cardiometabolic risk; cell type; crosslink; diabetic; disorder risk; epigenetic regulation; fetal; fetal programming; fetus cell; glucose uptake; in utero; maternal diabetes; novel; obesity risk; offspring; prenatal; programs; response; senescence; stem cell differentiation

Abstract Future risk for the development of cardiometabolic disease in youth, including type 2 diabetes, is increased by exposure to maternal diabetes (DM) in utero. These early exposures “program” the offspring toward cardiometabolic disease. Studies from my K23 indicate that a specific miRNA, miR-126, is highly abundant in human umbilical vein endothelial cells, placenta, and circulation, targeting elements in the insulin signaling pathway, potentially leading to insulin resistance. However, the effects of miR-126 on cell function and differences in mRNA targets based on cell type and diabetes exposure were not examined. My overall hypothesis is that maternal diabetes increases miR-126 expression in the infant, and this is an important epigenetic driver of insulin resistance and cellular metabolism. Therefore, the overarching goal of the proposed research is to determine how miR-126 disrupts cellular metabolism in fetal cells and to discover novel targets for these pathways. Perinatal studies of miRNA are limited in scope, with most focusing on candidate miRNA species and their putative targets without direct testing of the mechanistic impact. The current proposal will overcome these constraints and extend our current work by a) investigating the impact of maternal DM on cellular function, b) examining biological effects of miR-126 at the cellular level and c) identifying additional mRNA targets of miR-126 in a cell/context-specific fashion. Aim 1. To test the hypothesis that maternal diabetes exposure increases miR-126 abundance altering cellular metabolism. Aim 2. To test the hypothesis that the targets of miR-126 will be cell-type specific and altered by DM exposure. We hypothesize that DM exposure will result in decreased glucose uptake and proliferation, but an increase in senescence associated with increased abundance of miR-126; and in vitro, miR-126 will decrease glucose uptake in the adipocytes and decrease proliferation but increase senescence in the mesenchymal stem cell (MSC). Glucose uptake will be measured in adipocytes using glucose isotope. Proliferation will be assessed by MTT assay, and senescence will be assessed via flow cytometry in the MSCs. miR-126 will be transfected into the DM exposed and unexposed cells with repetition of the above studies to determine the direct effect of miR- 126. In the second aim, high-throughput sequencing of RNA by crosslinking immunoprecipitation (HITS-CLIP) in MSCs and differentiated adipocytes exposed and unexposed to DM will identify novel targets within each cell type as well as the impact of the DM exposure on target selection. These targets will then be examined by Western blot analysis. Understanding the biological and metabolic pathways altered by miRNAs will further elucidate their impact on cellular metabolism and increased risk of cardiometabolic complications, including obesity and diabetes, in youth exposed to DM. Understanding these alterations will be key to prevention.

抽象的 未来在包括2型糖尿病在内的青年中心脏代谢疾病发展的未来风险被增加 在子宫内暴露于产妇糖尿病（DM）。这些早期的暴露“计划”后代朝向 心脏代谢疾病。来自我的K23的研究表明，特定的miRNA miRNA，高度丰富 人脐静脉内皮细胞，胎盘和循环，靶向胰岛素信号中的元素 途径，可能导致胰岛素抵抗。但是，miR-126对细胞功能和细胞功能的影响 未检查基于细胞类型和糖尿病暴露的mRNA靶标的差异。我的整体 假设是遗传糖尿病会增加婴儿中的miR-126表达，这是一个重要的 胰岛素抵抗和细胞代谢的表观遗传驱动器。因此， 拟议的研究是确定miR-126如何破坏胎儿细胞中的细胞代谢并发现 这些途径的新目标。 miRNA的围产期研究的范围有限，大多数关注 候选miRNA物种及其推定目标，而无需直接测试机械影响。这 当前的建议将克服这些限制，并通过a）调查的影响来扩展我们当前的工作 母体DM对细胞功能，b）检查miR-126在细胞水平和c上的生物学作用） 以特定于细胞/上下文的方式识别miR-126的其他mRNA靶标。 目的1。测试材料糖尿病暴露会增加miR-126的丰度改变的假设 细胞代谢。 目的2。测试miR-126的靶标将是细胞类型的假设，并由DM改变 接触。 我们假设DM暴露会导致葡萄糖摄取和增殖减少，但增加 与miR-126的丰度增加相关的感应；在体外，miR-126将减少葡萄糖 吸收脂肪细胞并减少增殖，但会增加间充质干细胞的感受 （MSC）。葡萄糖摄取将在脂肪细胞中使用葡萄糖同位素进行测量。扩散将通过 MTT测定法和感应将通过MSC中的流式细胞仪评估。 mir-126将被翻译成 通过重复上述研究暴露的DM和意外细胞，以确定miR-的直接作用 126。在第二个目标中，通过交联免疫沉淀（hits-CLIP）对RNA进行高通量测序 在MSC和差异化的脂肪细胞中，暴露于DM的脂肪细胞将确定每个目标中的新目标 细胞类型以及DM暴露对目标选择的影响。然后将检查这些目标 Western印迹分析。了解miRNA改变的生物学和代谢途径将进一步 阐明它们对细胞代谢的影响，并增加心脏代谢并发症的风险，包括 肥胖和糖尿病，暴露于DM的青年中。了解这些改变将是预防的关键。