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MicroRNAs as effectors of variable human drug metabolism

MicroRNA 作为可变人类药物代谢的效应器

基本信息

批准号：
8827811
负责人：
Michael H Court
金额：
$ 28.21万
依托单位：
WASHINGTON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8827811
关键词：
3&apos Untranslated Regions Address Algorithms Alleles Bilirubin Binding Bioinformatics Biological Markers Biological Models Blood Cell Line Cells Clinical Code Complement Development Dose Drug usage Enzymes Gene Expression Gene Expression Profile Genes Genetic Polymorphism Genome Glucuronosyltransferase Goals HIV HIV Infections Hepatocyte Human Indium Individual Laboratories Lead Lentivirus Vector Libraries Liver Luciferases Malignant Neoplasms Messenger RNA Methods MicroRNAs Modeling Mutagenesis Mycophenolate Organ Patients Pharmaceutical Preparations Pharmacogenetics Pharmacogenomics Phenotype Post-Transcriptional Regulation Proteins Proteome Proteomics RNA Sequences RNA Splicing Regulation Reporter Response Elements Role Safety Sampling Small RNA Subfamily lentivirinae Testing Therapeutic Index Time UGT1A1 gene Untranslated RNA Variant Work cell type cost effective drug metabolism functional genomics genetic variant in vivo irinotecan mRNA Expression novel novel strategies overexpression prevent response transcription factor transcriptome sequencing transcriptomics

项目摘要

DESCRIPTION (provided by applicant): The promise of pharmacogenomics - identification of the right dose of the right drug the first time for everyone - has yet to be fully realized in part because we lack a complete understanding of the mechanisms underlying drug response variability. Recent studies suggest that microRNAs (miRs) - small noncoding RNAs that regulate gene expression post-transcriptionally - could be an important determinant of interindividual variability in drug metabolism and disposition. Here we propose to use a variety of novel approaches to identify miRs that are responsible for drug metabolizing enzyme variability. The focus of the application will be the UDP- glucuronosyltransferase (UGT) 1A enzymes that are critical to the elimination of many clinically important drugs with a narrow therapeutic index. Importantly, al 9 UGT1A enzymes (out of 18 total human UGTs) are encoded by spliced mRNAs that share an identical 3'-untranslated region (UTR). Although we have evidence that these enzymes are co-regulated by a post-transcriptional mechanism, virtually nothing is known regarding the role of miRs in UGT1A expression. Our overarching hypothesis is that specific miRs regulate UGT1A gene expression through the conserved UGT1A-3'-UTR mRNA region and/or indirectly by modulating regulatory transcription factor levels. To test this hypothesis, th following specific aims are proposed. In Aim 1 candidate miRs associated with UGT1A glucuronidation will be identified by multiple independent approaches, including bioinformatics, functional genomics screen, and whole genome (miR-transcriptome) association studies. The novel functional genomics screen we propose enables rapid identification of miRs that suppress a fluorescent- tagged miR 3'-UTR reporter from a pooled lentivirus library of all known human miRs. This method should have broad application outside of our field enabling a cost-effective rapid approach to describing of the "miR interactome". The reference UGT1A-3'-UTR allele will be evaluated as well as the major variant (*1b) associated with decreased bilirubin glucuronidation in vivo. miRs that are overexpressed in human liver bank samples with a low UGT1A activity and translational efficiency phenotype will also be identified by miR transcriptomics (arrays and Next gen small RNA-seq). In Aim 2 we wil confirm the functional impact of candidate miRs on UGT1A glucuronidation through overexpression (lenti-miRs) and knockdown (lenti- antagomiRs) of candidate miRs in human model cell lines and primary hepatocytes. In Aim 3 we will determine the mechanism of UGT1A regulation by functional miRs through use of UGT1A-3'UTR luciferase-reporter constructs and through studying effects of miR overexpression and knockdown in model cell lines and primary human hepatocytes on specific candidate transcription factor proteins (PXR and HNF1) as well as the entire proteome (iTRAQ LC-MS/MS method). This work will ultimately lead to development of validated predictive biomarkers (miR-associated polymorphisms and/or miR levels in blood) that would be incorporated into the pharmacogenetic algorithms currently being developed to guide dosing of low therapeutic index drugs.

描述（由申请人提供）：药物基因组学的承诺 - 第一次为每个人确定正确药物的正确剂量 - 尚未部分完全实现因为我们对药物反应变异性的机制缺乏完整的了解。最近的研究表明，微小RNA（miR）——转录后调节基因表达的小非编码RNA——可能是药物代谢和处置个体差异的重要决定因素。在这里，我们建议使用各种新方法来识别导致药物代谢酶变异的 miR。该应用的重点将是 UDP-葡萄糖醛酸基转移酶 (UGT) 1A 酶，该酶对于消除许多具有狭窄治疗指数的临床重要药物至关重要。重要的是，所有 9 种 UGT1A 酶（总共 18 种人类 UGT）均由共享相同 3'-非翻译区 (UTR) 的剪接 mRNA 编码。尽管我们有证据表明这些酶受到转录后机制的共同调节，但实际上对 miR 在 UGT1A 表达中的作用一无所知。我们的总体假设是，特定的 miR 通过保守的 UGT1A-3'-UTR mRNA 区域和/或通过调节调节转录因子水平间接调节 UGT1A 基因表达。为了检验这一假设，提出了以下具体目标。在目标 1 中，将通过多种独立方法来鉴定与 UGT1A 葡萄糖醛酸化相关的候选 miR，包括生物信息学、功能基因组学筛选和全基因组（miR-转录组）关联研究。我们提出的新型功能基因组学筛选能够从所有已知人类 miR 的慢病毒库中快速识别抑制荧光标记 miR 3'-UTR 报告基因的 miR。这种方法应该在我们的领域之外具有广泛的应用，从而能够以一种经济有效的快速方法来描述“miR相互作用组”。将评估参考 UGT1A-3'-UTR 等位基因以及与体内胆红素葡萄糖醛酸化减少相关的主要变异 (*1b)。在人肝库样本中过度表达且具有低 UGT1A 活性和翻译效率表型的 miR 也将通过 miR 转录组学（阵列和下一代小 RNA 测序）进行鉴定。在目标 2 中，我们将通过人模型细胞系和原代肝细胞中候选 miR 的过表达（lenti-miRs）和敲低（lenti-antagomiRs）来确认候选 miRs 对 UGT1A 葡萄糖醛酸化的功能影响。在目标 3 中，我们将通过使用 UGT1A-3'UTR 荧光素酶报告基因构建体，并通过研究模型细胞系和原代人肝细胞中 miR 过表达和敲低对特定候选转录因子蛋白（PXR 和 HNF1）以及整个蛋白质组（iTRAQ LC-MS/MS 方法）的影响，确定功能性 miR 调节 UGT1A 的机制。这项工作最终将导致开发经过验证的预测生物标志物（miR 相关多态性和/或血液中的 miR 水平），这些标志物将被纳入目前正在开发的药物遗传学算法中，以指导低治疗指数药物的剂量。

项目成果

期刊论文数量（17）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Relationship between the melanocortin-1 receptor (MC1R) variant R306ter and physiological responses to mechanical or thermal stimuli in Labrador Retriever dogs.

黑皮质素-1 受体 (MC1R) 变体 R306ter 与拉布拉多猎犬对机械或热刺激的生理反应之间的关系。

DOI：
10.1016/j.vaa.2016.05.003
发表时间：
2017
期刊：
Veterinary anaesthesia and analgesia
影响因子：
1.7
作者：
Perez,TaniaE;Mealey,KatrinaL;Burke,NealS;Grubb,TamaraL;Court,MichaelH;Greene,StephenA
通讯作者：
Greene,StephenA

Comparative metabolism of mycophenolic acid by glucuronic acid and glucose conjugation in human, dog, and cat liver microsomes.