title: A novel dual-fluorescence strategy for functionally validating microRNA targets in 3' untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212.
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 K(ir)2.1 [KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2)] which is dysregulated in cardiac and vascular disorders. The 3'UTR (untranslated region) 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. HEK (human embryonic kidney)-293 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 down-regulator of K(ir)2.1 expression, and was used to investigate the targeting of the K(ir)2.1 3'UTR by miR-212. The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 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.
authors.affiliation: Centre for Vision and Vascular Science, Queen's University of Belfast, Institute of Clinical Science, Block A, Royal Victoria Hospital, Grosvenor Road, Belfast, BT12 6BA, UK.
authors.forename: Dana
authors.initials: D
authors.lastname: Goldoni
authors.forename: Janet M
authors.initials: JM
authors.lastname: Yarham
authors.forename: Mary K
authors.initials: MK
authors.lastname: McGahon
authors.forename: Anna
authors.initials: A
authors.lastname: O'Connor
authors.forename: Jasenka
authors.initials: J
authors.lastname: Guduric-Fuchs
authors.forename: Kevin
authors.initials: K
authors.lastname: Edgar
authors.forename: Denise M
authors.initials: DM
authors.lastname: McDonald
authors.forename: David A
authors.initials: DA
authors.lastname: Simpson
authors.forename: Anthony
authors.initials: A
authors.lastname: Collins
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D020029:Base Pairing; D001665:Binding Sites; D015536:Down-Regulation; D005470:Fluorometry / Q000379:methods; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D035683:MicroRNAs / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D020413:3' Untranslated Regions; D049452:Green Fluorescent Proteins; D008156:Luciferases; D008164:Luminescent Proteins; D035683:MicroRNAs; D024661:Potassium Channels, Inwardly Rectifying; D000097573:Red Fluorescent Protein; C489457:KCNJ2 protein, human; C551411:MIRN212 microRNA, human; C485184:enhanced green fluorescent protein