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

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 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.