MicroRNAs (miRNAs) are a class of noncoding small RNAs modulating gene expression. By annealing to the 3’ terminal untranslated region (3’UTR) of the target mRNA, they lead to translational repression. K_ir2.1 is a member of the gene subfamily coding for inward rectifier potassium channels. K_ir2.1 passes inwardly rectifying K⁺ current (I_K1) which plays an important role in repolarising and stabilizing the membrane potential in cardiac myocytes; indeed down-regulation of inward rectifier current occurs in cardiomyopathy and is proarrhythmic (1). Because the causes of I_K1 reduction are still unknown and because a variation in expression of miRNAs has been shown in cardiac hypertrophy and heart failure (2-4) we hypothesize that miRNAs are involved in the down-regulation of K_ir2.1 and I_K1. We aim to demonstrate the relationship between miRNAs and down-regulation of K_ir2.1 by identifying miRNAs that are up-regulated in cardiomyopathy (2-4) and predicted to target the K_ir2.1 3’UTR (TargetScan; Miranda; PicTar), transfecting them into HEK293 cells and assaying for functional targeting using a luciferase reporter with putative target sites in the 3’UTR. miR-1 was chosen as control and to validate the method as it has been shown to target K_ir2.1 (5). Two luciferase reporter plasmids were constructed from pMIR-REPORT (Applied Biosystems); one with the K_ir2.1 3’UTR miR-1 target site (pLuc2.1-1), and the other with predicted miR-132, miR-212 and miR-320 target sites (pLuc2.1-3). MiRNA expression plasmids were produced in the siRNA expression vector pSM30 (provided by Dr. G. Du, UTHSC, Houston) by inserting oligonucleotide cassettes of mature miRNA sequences. HEK293 cells were transfected with a luciferase reporter plasmid, miRNA vector and a β-galactosidase plasmid as normalizer. After 48 hours the Dual-Light® assay (Applied Biosystems) was performed. The method has been optimized with miR-1 and pLuc2.1-1. Luciferase/β-galactosidase was 40-44% lower in cells transfected with miR-1 vs pSM30 with no insert (p<0.01, n=3, t-test, repeated three times with different plasmid ratios). miR-212 down-regulated functional expression of pLuc2.1-3 in comparison with miR-1, which is a negative control for pLuc2.1-3. Luciferase/β-galactosidase was 72% lower in cells transfected with miR-212 vs miR-1 (p<0.05, n=3, t-test). In summary, we have demonstrated miRNA targeting using the pSM30 vector and provided evidence for a miR-212 target in the 3’UTR of K_ir2.1. These studies are intended to form the basis of future studies aimed at demonstrating down-regulation of I_K1 by specific miRNAs.