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

基本信息

批准号：
10380690
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
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10380690
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

项目摘要

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.

抽象的青少年未来患心脏代谢疾病（包括 2 型糖尿病）的风险会增加在子宫内接触母亲糖尿病 (DM) 这些早期接触“编程”了后代。我的 K23 研究表明，一种特定的 miRNA，miR-126，在心脏代谢疾病中含量很高。人脐静脉内皮细胞、胎盘和循环，靶向胰岛素信号传导元件然而，miR-126 对细胞功能和胰岛素抵抗的影响。未检查基于细胞类型和糖尿病暴露的 mRNA 靶点差异。假设母亲糖尿病会增加婴儿中 miR-126 的表达，这是一个重要的因此，胰岛素抵抗和细胞代谢的表观遗传驱动因素。拟议的研究旨在确定 miR-126 如何扰乱胎儿细胞的细胞代谢并发现这些途径的围产期研究的新靶标范围有限，大多数集中于候选 miRNA 种类及其假定目标，而无需直接测试机械影响。当前的提案将克服这些限制，并通过以下方式扩展我们当前的工作：a) 调查以下因素的影响母体 DM 对细胞功能的影响，b) 在细胞水平检查 miR-126 的生物学效应，c) 以细胞/环境特异性方式识别 miR-126 的其他 mRNA 靶标。目标 1. 检验母亲患糖尿病会增加 miR-126 丰度并改变这一假设细胞代谢。目标 2. 检验 miR-126 的靶标具有细胞类型特异性并会被 DM 改变的假设接触。我们发现，DM 暴露会导致葡萄糖摄取和增殖减少，但会增加衰老与 miR-126 丰度增加相关；在体外，miR-126 会降低血糖；脂肪细胞的摄取并减少增殖，但增加间充质干细胞的衰老 (MSC)。将使用葡萄糖同位素测量脂肪细胞中的葡萄糖摄取，并评估其增殖。 MTT 测定，并通过流式细胞术评估转染 miR-126 的 MSC 的衰老情况。 DM暴露和未暴露的细胞重复上述研究以确定miR- 126. 第二个目标，通过交联免疫沉淀对RNA进行高通量测序（HITS-CLIP）在暴露和未暴露于 DM 的 MSC 和分化脂肪细胞中，将识别每个细胞中的新靶点然后将检查细胞类型以及 DM 暴露对目标选择的影响。蛋白质印迹分析将进一步了解 miRNA 的生物学和代谢途径。阐明它们对细胞代谢的影响和增加心脏代谢并发症的风险，包括在接触糖尿病的青少年中，了解这些变化将是预防的关键。