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.
Data(s) |
18/10/2012
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Resumo |
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. |
Identificador | |
Idioma(s) |
eng |
Direitos |
info:eu-repo/semantics/restrictedAccess |
Fonte |
Goldoni , D , Yarham , J , McGahon , M , O'Connor , A , Guduric-Fuchs , J , Edgar , K , McDonald , D , Simpson , D & Collins , T 2012 , ' 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. ' Biochemical Journal , vol 448 , no. 1 , pp. 103-113 . DOI: 10.1042/BJ20120578 |
Palavras-Chave | #patch clamp #image analysis #HeLa #HEK293 #microRNA #/dk/atira/pure/subjectarea/asjc/1300/1303 #Biochemistry #/dk/atira/pure/subjectarea/asjc/1300/1307 #Cell Biology #/dk/atira/pure/subjectarea/asjc/1300/1312 #Molecular Biology |
Tipo |
article |