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

 描述（由申请方提供）：在人类寿命期间，药物代谢酶的发育变化导致许多药物的疗效和安全性较差;然而，导致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microRNAs（miRNAs）的发育变化可能有助于药物代谢基因和上游调控基因的这些发育变化。许多这些药物处置基因的表达与胎儿、儿童和成人发育期肝脏miRNA的变化呈负相关。这些负相关性也与预测和验证的miRNA-mRNA相互作用相结合。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控基因的表达，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，在本奖学金申请中，我们将通过测量HepaRG细胞中mRNA、蛋白质和蛋白质活性的变化来验证我们所有的miRNA-mRNA预测，从而验证这一假设。将使用qPCR测量mRNA水平的表达，使用蛋白质印迹法测量蛋白质，使用药物探针测量蛋白质活性，其中可以测量母体/代谢产物药物浓度并在实验组和对照组之间进行比较。将通过使用CRIPSR/Cas9核酸酶RNA指导的基因组编辑系统比较含有插入基因组中的种子序列的任一变体的HepaRG细胞来测试SNP改变miRNA调控的效果。使用该系统，我们还能够去除种子序列，以验证种子序列是其靶向mRNA的3 'UTR的关键结合区域。将使用qPCR测量两种变体之间或种子序列与种子序列敲除之间的mRNA水平变化。经过验证可以改变体外miRNA调节的SNP将通过回顾性基因分型健康志愿者的DNA样本进行体内测试，这些志愿者已经使用细胞色素P450特异性药物探针表征了其肝脏代谢活性。这些研究的结果将提高我们对发育中肝脏中药物代谢和反应的miRNA调节的理解。这将增加我们对发展药理学驱动机制的理解，并确定可用于更好地预测药物反应和个性化治疗的遗传变异。

 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.