A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Dana Goldoni, Janet Yarham, Mary McGahon, Anna O'Connor, Jasenka Guduric-Fuchs, Kevin Edgar, Denise McDonald, David Simpson, Tony Collins

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.
Original languageEnglish
Pages (from-to)103-113
Number of pages11
JournalBiochemical Journal
Volume448
Issue number1
Early online date13 Aug 2012
DOIs
Publication statusPublished - 18 Oct 2012

Keywords

  • patch clamp
  • image analysis
  • HeLa
  • HEK293
  • microRNA

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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