Circulating miRNAs and prediction of beta-cell treatment response: The Restoring Insulin Secretion Study

摘要 尽管进行了治疗，但仍有相当比例的2型糖尿病（T2 D）患者未出现临床症状。 改进（即，缺乏治疗反应）。在对恢复胰岛素分泌（RISE）数据的分析中， 研究，一项随机对照试验，测试药物和手术治疗改善和 维持β细胞功能，我们团队的成员已经证明了治疗的实质性异质性 使用来自高血糖钳夹的金标准测量在β细胞水平的反应。我们所知甚少， 然而，关于T2 D治疗反应异质性的影响因素，缺乏前瞻性的工具， 识别治疗应答者和非应答者。其主要原因是缺乏循环生物标志物， 预测或反映β细胞水平的治疗反应。葡萄糖重要基因的表观遗传调控 和胰岛素代谢的短非编码RNA，如microRNA（miRNA）可能介导改善或 β细胞功能在治疗中得到保护，循环miRNA可能是治疗的预测性生物标志物 反应在β细胞中，通过miRNAs调控基因表达对胰岛素分泌和细胞增殖都至关重要。 周转此外，分泌到循环中的miRNAs可能反映了组织水平的表达差异， 和/或主动参与器官间串扰。虽然在各种组织和循环中的miRNAs 在人体或体外模型中暴露于RISE干预措施后， β细胞中的大多数miRNAs尚未被描述。使用来自RISE儿科的数据和样本 药物研究（n=49; R 03 DK 122100; PI Wander），我们鉴定了9种血浆miRNA， 与10-19岁青年的治疗反应相关，包括两种新的miRNA，miR-4468和miR-6727， 其被预测通过Bcl-2样1和胰岛淀粉样多肽调节胰岛细胞凋亡。我们的目标是 表征与成人RISE中β细胞功能的保存或改善相关的miRNA 受试者，通过钳夹衍生的β细胞反应测量与胰岛素敏感性配对，治疗后 用药物或胃束带为了实现这一目标，我们提出了一个两部分的研究。在目标1中，我们将分析 在巢式病例对照中使用下一代测序在基线和治疗后检测循环miRNA RISE中治疗应答者与非应答者的样本。在目标2中，我们将检查miR-4468、miR-2468和miR-2468对细胞增殖的影响。 6727，以及Aim 1中鉴定的miRNA对β细胞功能、存活和功能获得性去分化的影响。 在人类胰岛中使用基于锁核酸的miRNA模拟物的体外实验。我们的首要假设是 循环miRNA通过影响胰岛素分泌和β细胞存活而导致治疗异质性。 据我们所知，该项目将是第一个直接测试治疗相关的miRNA在介导或 反映治疗反应。更好地理解miRNAs作为治疗异质性的生物标志物， 通过前瞻性识别可能从特定治疗中受益的亚组来促进T2 D精准医疗 并促进有针对性的干预措施的发展，以预防或延缓T2 D的进展。

Abstract Despite treatment, a significant proportion of patients with type 2 diabetes (T2D) do not experience clinical improvement (i.e., lack treatment response). In analyses of data from the Restoring Insulin Secretion (RISE) Study, a randomized controlled trial testing the potential of medical and surgical therapies to improve and maintain β-cell function, members of our team have demonstrated substantial heterogeneity in treatment response at the level of the β cell using gold standard measures from hyperglycemic clamps. We know little, however, about contributing factors to heterogeneity of T2D treatment response and lack tools to prospectively identify treatment responders and non-responders. A major reason for this is lack of circulating biomarkers that predict or reflect treatment response at the level of the β cell. Epigenetic regulation of genes important in glucose and insulin metabolism by short non-coding RNAs such as microRNAs (miRNAs) may mediate improvement or preservation of β-cell function with treatment, and circulating miRNAs may be predictive biomarkers of treatment response. In the β cell, regulation of gene expression by miRNAs is crucial to both insulin secretion and cell turnover. In addition, miRNAs secreted into the circulation may reflect tissue-level differences in expression and/or participate actively in inter-organ crosstalk. While miRNAs in a variety of tissues and in the circulation have been shown to be altered by exposure to the RISE interventions in humans or in vitro models, effects of most of these miRNAs in the β cell are not yet described. Using data and samples from the RISE Pediatric Medication Study (n=49; R03DK122100; PI Wander), we identified 9 plasma miRNAs that are prospectively related to treatment response in youth aged 10–19 years including two novel miRNAs, miR-4468 and miR-6727, which are predicted to regulate islet apoptosis via Bcl-2 like 1 and islet amyloid polypeptide. Our objective is to characterize miRNAs that are related to preservation or improvement in β-cell function among adult RISE participants, defined by clamp-derived measures of β-cell response paired with insulin sensitivity, after treatment with medications or gastric banding. To achieve this goal, we propose a two-part study. In Aim 1, we will assay circulating miRNAs using next-generation sequencing at baseline and after treatment in a nested case-control sample of treatment responders vs. non-responders in RISE. In Aim 2, we will examine effects of miR-4468, miR- 6727, and miRNAs identified in Aim 1 on β-cell function, survival, and de-differentiation in gain-of-function in vitro experiments using locked nucleic acid-based miRNA mimics in human islets. Our overarching hypothesis is that circulating miRNAs contribute to treatment heterogeneity via effects on insulin secretion and β-cell survival. To our knowledge, this project will be the first to directly test the role of treatment-related miRNAs in mediating or reflecting treatment response. Better understanding of miRNAs as biomarkers of treatment heterogeneity will facilitate T2D precision medicine by prospectively identifying subgroups likely to benefit from specific treatments and promote development of targeted interventions to prevent or delay the progression of T2D.