Age-related changes in microRNA expression effect on drug metabolism in human liver

年龄相关的 microRNA 表达变化对人肝脏药物代谢的影响

• 批准号: 9269898
• 负责人: Kimberly S. Collins
• 金额: $ 1.06万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9269898
• 关键词: 3' Untranslated Regions; Address; Adult; Affect; Age; Aging; Automobile Driving; Binding; Binding Sites; CRISPR/Cas technology; Cells; Childhood; Clinical; Complex; Control Groups; Coupled; Cytochrome P450; DNA; Data; Development; Dose; Drug Binding Site; Drug Kinetics; Drug Regulations; Drug Targeting; Drug usage; Emerging Technologies; Enzymes; Fellowship; Gene Targeting; Genes; Genetic Transcription; Genetic Variation; Genome; Genotype; Guide RNA; Hepatic; Hepatocyte; Human; In Vitro; Individual; Knock-out; Knowledge; Label; Lead; Length; Liver; Longevity; Measures; Messenger RNA; Metabolic; MicroRNAs; Nucleotides; Parents; Pediatrics; Pharmaceutical Preparations; Pharmacogenomics; Pharmacology; Population; Prediction of Response to Therapy; Proteins; Reaction; Regulation; Regulator Genes; Ribonucleases; Safety; Sampling; Seeds; Site; System; Testing; Translating; Translations; Untranslated RNA; Untranslated Regions; Variant; Western Blotting; age related; base; clinically relevant; drug efficacy; drug metabolism; fetal; genetic variant; genome editing; healthy volunteer; improved; in vivo; innovation; mRNA Expression; novel; public health relevance; response

 DESCRIPTION (provided by applicant): Developmental changes in drug metabolizing enzymes during the human lifespan pose contribute to the poor efficacy and safety of many drugs; however, the mechanisms responsible for these changes are unknown. We have strong preliminary data suggesting that developmental changes in hepatic microRNAs (miRNAs) may contribute to these developmental changes in drug metabolism genes and upstream regulatory genes. Expression of many of these drug disposition genes negatively correlate with changes in hepatic miRNA across the fetal, pediatric, and adult developmental periods. These negative correlations are also coupled with both predicted and validated miRNA-mRNA interactions. There is also evidence that genetic variants in the 3'UTR (untranslated region), have the ability to affect drug metabolism by creating or abolishing miRNA binding sites. We hypothesize that hepatic miRNAs post-transcriptionally regulate expression of drug metabolizing genes and upstream regulatory genes, and that SNPs in miRNA binding sites within these genes alter miRNA ability repress translation. Therefore in this fellowship application we will test this hypothesis by validating all of our miRNA-mRNA predictions by measuring changes in mRNA, protein, and protein activity in HepaRG cells when transfected with miRNAs predicted to target the gene in question. Expression of mRNA levels will be measured using qPCR, protein using western blots, and protein activity using drug probes in which the parent/metabolite drug concentrations can be measured and compared between the experimental and control groups. The effect of SNPs altering miRNA regulation will be tested by comparing HepaRG cells containing either variation of the seed sequence inserted into the genome using the CRIPSR/Cas9 nuclease RNA-guided genome editing system. With this system, we are also able to remove the seed sequence in order to verify that the seed sequence is the critical binding region of the 3'UTR of its targeted mRNA. Changes in mRNA levels will be measured using qPCR between the two variants or the seed sequence versus the seed sequence knockout. SNPs validated to alter miRNA regulation in vitro, will then be tested in vivo by retrospectively genotyping DNA samples from healthy volunteers that already have their hepatic metabolic activity characterized using cytochrome P450 specific drug probes. The results from these studies will improve our understanding of the miRNA regulation of drug metabolism and response in the developing liver. This will increase our understanding the mechanism driving developmental pharmacology as well as identify genetic variations that can be used to better predict drug response and individualize therapies.

 描述（由申请人提供）：人类生命周期中药物代谢酶的发育变化导致许多药物的功效和安全性较差；然而，造成这些变化的机制尚不清楚。我们有强有力的初步数据表明，肝脏 microRNA (miRNA) 的发育变化可能导致药物代谢基因和上游调控基因的发育变化。许多药物处置基因的表达与胎儿、儿童和成人发育时期肝脏 miRNA 的变化呈负相关。这些负相关性还与预测和验证的 miRNA-mRNA 相互作用相关。还有证据表明 3'UTR（非翻译区）中的遗传变异能够通过创建或消除 miRNA 结合位点来影响药物代谢。我们假设肝脏 miRNA 转录后调节药物代谢基因和上游调节基因的表达，并且这些基因内 miRNA 结合位点的 SNP 改变 miRNA 抑制翻译的能力。因此，在本次奖学金申请中，我们将通过测量 HepaRG 细胞转染预测针对相关基因的 miRNA 时 mRNA、蛋白质和蛋白质活性的变化来验证我们所有 miRNA-mRNA 预测，从而检验这一假设。将使用 qPCR 测量 mRNA 水平的表达，使用蛋白质印迹测量蛋白质，并使用药物探针测量蛋白质活性，其中可以测量母体/代谢物药物浓度并在实验组和对照组之间进行比较。通过使用 CRIPSR/Cas9 核酸酶 RNA 引导的基因组编辑系统插入基因组中的任一种子序列变异的 HepaRG 细胞进行比较，可以测试 SNP 改变 miRNA 调节的效果。通过该系统，我们还能够去除种子序列，以验证种子序列是其目标 mRNA 3'UTR 的关键结合区域。将使用两个变体之间或种子序列与种子序列敲除之间的 qPCR 来测量 mRNA 水平的变化。在体外验证的 SNP 可以改变 miRNA 调节，然后通过对来自健康志愿者的 DNA 样本进行回顾性基因分型进行体内测试，这些志愿者已经使用细胞色素 P450 特异性药物探针对其肝脏代谢活性进行了表征。这些研究的结果将提高我们对 miRNA 对药物代谢和发育中肝脏反应的调节的理解。这将增加我们对驱动发育药理学机制的理解，并确定可用于更好地预测药物反应和个体化治疗的遗传变异。

项目成果

Age-Related Changes in MicroRNA Expression and Pharmacogenes in Human Liver.

• DOI: 10.1002/cpt.145
• 发表时间: 2015-08
• 期刊: Clinical pharmacology and therapeutics
• 影响因子: 6.7
• 作者: Burgess KS;Philips S;Benson EA;Desta Z;Gaedigk A;Gaedigk R;Segar MW;Liu Y;Skaar TC
• 通讯作者: Skaar TC