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

摘要 年轻人患心脏代谢性疾病，包括2型糖尿病的未来风险增加 在宫内暴露于母体糖尿病(DM)。这些早期的暴露将子代“编程”为 心脏代谢疾病。来自我的K23的研究表明，一种特定的miRNA，miR-126，在 人脐静脉内皮细胞、胎盘和循环，胰岛素信号中的靶向元件 途径，可能导致胰岛素抵抗。然而，miR-126对细胞功能和 基于细胞类型和糖尿病暴露的信使核糖核酸靶标的差异没有被检查。我的整体 假设是母亲糖尿病增加了婴儿miR-126的表达，这是一个重要的 胰岛素抵抗和细胞代谢的表观遗传驱动因素。因此，该计划的首要目标是 拟议的研究是确定miR-126如何扰乱胎儿细胞的细胞新陈代谢，并发现 这些途径的新靶点。围产期对miRNA的研究范围有限，大多数集中在 候选miRNA物种及其假定的目标，而不直接测试机械影响。这个 目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 母体糖尿病对细胞功能的影响，b)在细胞水平上检查miR-126的生物学效应，以及c) 以细胞/上下文特定的方式识别miR-126的其他信使核糖核酸靶点。 目的1.检验母亲糖尿病暴露增加miR-126丰度改变的假设 细胞新陈代谢。 目的2.验证假设miR-126的靶点将是细胞类型特异性的并被DM改变 曝光。 我们假设，接触DM会导致葡萄糖摄取和增殖减少，但 衰老与miR-126丰度增加有关；在体外，miR-126会降低血糖 脂肪细胞的摄取和间充质干细胞的抑制增殖但加速衰老 (MSC)。脂肪细胞的葡萄糖摄取量将使用葡萄糖同位素进行测量。扩散将通过以下方式进行评估 四甲基偶氮唑盐比色法，并通过流式细胞仪检测MSCs的衰老。MIR-126将被转化为 对DM暴露和未暴露的细胞进行重复上述研究，以确定miR-1的直接作用。 126.第二个目标是用交联型免疫沉淀法(HITS-CLIP)进行高通量的RNA测序 在MSCs和分化的脂肪细胞中，暴露和未暴露于DM的细胞将识别每个细胞内的新靶点 细胞类型以及DM暴露对目标选择的影响。然后，这些目标将由 蛋白质印迹分析。了解miRNAs改变的生物和代谢途径将进一步 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 肥胖和糖尿病，在青年中暴露于DM。了解这些变化将是预防的关键。

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.