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

摘要 年轻人未来患心脏代谢疾病（包括2型糖尿病）的风险增加， 在子宫内暴露于母体糖尿病（DM）。这些早期的暴露“程序”的后代， 心脏代谢疾病来自我的K23的研究表明，一种特定的miRNA，miR-126，在细胞中高度丰富， 人脐静脉内皮细胞、胎盘和循环，胰岛素信号传导中的靶向元件 途径，可能导致胰岛素抵抗。然而，miR-126对细胞功能的影响和 没有检查基于细胞类型和糖尿病暴露的mRNA靶的差异。我的整体 一种假说是母亲糖尿病增加了婴儿中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， 拟议的研究是确定miR-126如何破坏胎儿细胞的细胞代谢，并发现 这些途径的新目标。围产期miRNA的研究范围有限，大多数集中在 候选miRNA种类及其推定的靶点，而不直接测试机制影响。的 目前的建议将克服这些限制，并通过以下方式扩展我们目前的工作：a）调查 母体DM对细胞功能的影响，B）检查miR-126在细胞水平上的生物学作用，和c） 以细胞/环境特异性方式鉴定miR-126的其他mRNA靶标。 目标1。为了检验母体糖尿病暴露增加miR-126丰度改变的假设， 细胞代谢 目标2.检验miR-126的靶点具有细胞类型特异性并被DM改变的假设 exposure. 我们假设DM暴露将导致葡萄糖摄取和增殖减少，但增加了细胞增殖。 衰老与miR-126丰度增加相关;在体外，miR-126将降低葡萄糖 在脂肪细胞中的摄取，并减少间充质干细胞的增殖，但增加衰老 （MSC）。将使用葡萄糖同位素在脂肪细胞中测量葡萄糖摄取。扩散将通过以下方式进行评估： MTT测定，并通过流式细胞术在MSC中评估衰老。将miR-126转染入 DM暴露和未暴露的细胞，重复上述研究以确定miR- 126.第二个目标是通过交联免疫沉淀（HITS-CLIP）进行RNA高通量测序 在暴露于和未暴露于DM的MSC和分化的脂肪细胞中， 细胞类型以及DM暴露对靶选择的影响。这些目标将由 Western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心脏代谢并发症风险的增加，包括 肥胖和糖尿病，在青年暴露于糖尿病。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.