In Vitro Validation of miRNA‐Mediated Gene Expression Linked to Drug Metabolism

Botros B. Shenoda1, Sujay Ramanathan1, Seena K. Ajit1

1 Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 9.26
DOI:  10.1002/cpph.30
Online Posting Date:  December, 2017
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Pharmacogenomic approaches used to investigate how genes affect drug responses are critical for designing personalized therapies aimed at maximizing efficacy and minimizing adverse effects. Drug efficacy is often dependent on the sequence and expression levels of drug target genes or those involved in the metabolism and transport of the therapeutic agent. Expression of these genes, in turn, is negatively regulated by small noncoding miRNAs. The levels of miRNAs in bodily fluids have been studied extensively as potential diagnostic and prognostic biomarkers. Studies have shown that miRNAs regulate multiple genes and sequence homology is used to predict which genes are subject to regulation by a particular miRNA. Once a gene is identified as a potential target for an miRNA of interest, experiments are undertaken to confirm that the miRNA interacts with the target gene and can alter its level of expression and/or its activity. For example, the differential expression of miRNAs in whole blood obtained from good and poor responders to ketamine has been reported both prior to, and following treatment for complex regional pain syndrome. In this case, hsa‐miR‐548d‐5p was significantly lower in poor responders relative to good responders. This miRNA was predicted to target UDP‐glucuronyl transferase 1A1 (UGT1A1), a key drug metabolizing enzyme. Described in this unit are protocols used to confirm miR‐548d‐5p‐mediated UGT1A1 regulation. The approaches described can be employed broadly for the validation of miRNA‐mediated negative regulation of any gene. Determining miRNA‐mediated regulation of enzymes and transporters affecting drug metabolism is a critical step in designing personalized therapy and for understanding the mechanisms responsible for variations in the responses to therapeutic agents. © 2017 by John Wiley & Sons, Inc.

Keywords: drug metabolism; MicroRNA; ketamine; Hsa‐miR‐548d‐5p; UDP‐glucuronyl transferase; glucuronidation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Confirm Binding of miR‐548d‐5p to 3′ UTR of UGT1A1 Using Luciferase Reporter Assay
  • Basic Protocol 2: Confirm the Role of miRNA in Regulating the Expression Levels of Target Gene mRNA
  • Basic Protocol 3: Functional Assay to Validate the Regulatory Role for miRNA
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Confirm Binding of miR‐548d‐5p to 3′ UTR of UGT1A1 Using Luciferase Reporter Assay

  • HEK‐293 cells (ATCC #CRL‐1573)
  • Trypsin/EDTA solution (Sigma, cat. no. T3924)
  • Complete cell culture medium [DMEM (Sigma, cat. no. D5796) with 10% fetal bovine serum (FBS; Corning, cat. no. 35‐010‐CV) and 1% Penicillin streptomycin (Cellgro, cat. no. 30‐002‐CI)]
  • Lipofectamine 2000 (Invitrogen, cat. no. 11668019)
  • Luc‐Pair™ Duo‐Luciferase Assay Kit 2.0 (GeneCopoeia, cat. no. LPFR‐P010)
  • Precursor miRNA clone (e.g., hsa‐mir‐548d Precursor miRNA clone; GeneCopoeia, cat. no. HmiR0132)
  • 3′ UTR clones (e.g., human UGT1A1; GeneCopoeia, cat. no. HmiT013615‐MT01)
  • miRNA target control clone vector (GeneCopoeia, cat. no. CmiT000001‐MT05)
  • Phosphate‐buffered saline (PBS; Lonza, cat. no. 17‐517Q)
  • DMEM (Sigma, cat. no. D5796)
  • Fetal bovine serum (FBS; Corning, cat. no. 35‐010‐CV)
  • 75‐cm2 flask
  • 15‐ml conical tubes
  • Centrifuge
  • Hemacytometer
  • 96‐well cell culture plates
  • 37°C incubator
  • Rocking platform or orbital shaker
  • GloMax®‐96 Microplate Luminometer (Promega)

Basic Protocol 2: Confirm the Role of miRNA in Regulating the Expression Levels of Target Gene mRNA

  • HepG2 cells (ATCC® #HB‐8065™)
  • Eagle's Minimum Essential Medium (EMEM; ATCC, cat. no. 30‐2003)
  • Penicillin/streptomycin (Corning, cat. no. 30‐002‐CI)
  • Precursor miRNA clone (e.g., hsa‐mir‐548d Precursor miRNA clone GeneCopoeia, cat. no. HmiR0132)
  • miRNA scrambled control clone (GeneCopoeia, cat. no. CmiR0001)
  • Xtreme gene HP DNA transfection reagent (Roche, cat. no. 06366244001)
  • Phosphate‐buffered saline (PBS; Corning, cat. no. 21‐040)
  • RNase decontamination solution (e.g., Ambion RNaseZap® Solution) or RNase Zap wipes (Ambion, cat. no. AM9786 and AM9788)
  • 0.25% Trypsin‐EDTA solution (Sigma, cat. no. T4049)
  • mirVana miRNA isolation kit (Life Technologies, cat. no. AM1561)
  • Ice
  • Acid Phenol:Chloroform (Ambion, cat. no. AM9722)
  • Ethanol 200 Proof for molecular biology (Decon Laboratories, cat. no. 3916EA)
  • RNase‐free DNase set (Qiagen, cat. no. 79254)
  • Water (nuclease‐free) (Ambion, cat. no. AM9937)
  • Maxima First‐Strand cDNA Synthesis Kit (Thermo Scientific, cat. no. K1642)
  • Taqman Fast Universal PCR master mix (Life Technologies, cat. no. 4352042)
  • Taqman gene expression assay mix for target gene (e.g., UGT1A1 Life Technologies, cat. no. Hs02511055_s1)
  • Taqman gene expression assay mix for housekeeping mRNA (e.g., 18sRNA Life Technologies, cat. no. 4319413E)
  • 6‐well plates
  • RNase‐free barrier pipette tips (for transferring solutions)
  • Cell scrapers
  • Centrifuge
  • Vortex mixer
  • Microcentrifuge tubes
  • Filter cartridge (Invitrogen, cat. no. 10051G2)
  • Nanodrop Spectrophotometer
  • Thermal cycler
  • MicroAmp fast optical 96‐well plate reaction plate (Life Technologies, cat. no. 4344906)
  • Optical adhesive films (Life Technologies, cat. no. 4311971)
  • 7900HT Fast Real‐Time PCR System (Applied Biosystem)

Basic Protocol 3: Functional Assay to Validate the Regulatory Role for miRNA

  • 10 mM Tris·Cl, pH 7.4 (Teknova, cat. no. T3456)
  • EDTA (Fermentas, cat. no. R1021)
  • Glycerol (Sigma‐Aldrich, cat. no. G5516)
  • 50 mM potassium phosphate buffer, pH 7.4 (Sivertsson et al., )
  • Bio‐Rad DC™ Protein Assay Kit I (BIO‐RAD, cat. no. 5000111)
  • Albumin from bovine serum (Sigma‐Aldrich, cat. no. A2934)
  • UGT‐Glo™ Assay kit (Promega, cat. no. V2081)
  • Ultracentrifuge tubes
  • Sonicator
  • Centrifuge
  • White opaque 96‐well plates
  • 37°C incubator
  • GloMax R‐96 microplate Luminometer (Promega)
  • Additional reagents and solutions for transfecting HepG2 cells (see protocol 2)
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Internet Resources
  Bio‐Rad DC Protein Assay protocol:‐
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