title: Regulation of heme oxygenase-1 protein expression by miR-377 in combination with miR-217.
abstract: Heme oxygenase-1 (HO-1) enzyme plays a critical role in metabolizing the excess heme generated during hemolysis. Our previous studies suggested that during intravascular hemolysis the expression of HO-1 protein is not sufficient to reduce the oxidative burden of free heme in the vasculature. This led us to hypothesize that a post-translational mechanism of control exists for HO-1 expression. Micro-RNAs (miRNA) affect gene expression by post-transcriptional gene regulation of transcripts. We performed in silico analysis for the human HMOX1-3' untranslated region (3' UTR) and identified candidate miRNA binding sites. Two candidate miRNAs, miR-377 and miR-217, were cloned and co-transfected with a luciferase vector containing the human HMOX1-3'UTR region. The combination of miR-377 and miR-217 produced a 58% reduction in HMOX1-3'UTR luciferase reporter expression compared with controls. The same constructs were then used to assess how overexpression of miR-217 and miR-377 affected HO-1 levels after induction with hemin. Cells transfected with the combination of miR-377 and miR-217 exhibited no change in HMOX1 mRNA levels, but a significant reduction in HMOX1 (HO-1) protein expression and enzyme activity compared with non-transfected hemin-stimulated controls. Transfection with either miR-377 or miR-217 alone did not produce a significant decrease in HO-1 protein expression or enzyme activity. Knockdown of miR-217 and miR-377 in combination leads to up-regulation of HO-1 protein. Exposure to hemin induced a significant reduction in miR-217 expression and a trend toward decreased miR-377 expression in two different cells lines. In summary, these data suggests that the combination of miR-377 and miR-217 help regulate HO-1 protein expression in the presence of hemin.
authors.affiliation: Department of Medicine, Division of Hematology, Oncology, and Transplantation, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
authors.forename: Joan D
authors.initials: JD
authors.lastname: Beckman
authors.forename: Chunseng
authors.initials: C
authors.lastname: Chen
authors.forename: Julia
authors.initials: J
authors.lastname: Nguyen
authors.forename: Venugopal
authors.initials: V
authors.lastname: Thayanithy
authors.forename: Subbaya
authors.initials: S
authors.lastname: Subramanian
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Steer
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Vercellotti
mesh_terms: D004357:Drug Synergism; D004359:Drug Therapy, Combination; D005786:Gene Expression Regulation / Q000187:drug effects*; D051547:Heme Oxygenase-1 / Q000096:biosynthesis / Q000235:genetics*; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000494:pharmacology; D014162:Transfection
chemical_list: C553315:MIRN217 microRNA, human; C534063:MIRN377 microRNA, human; D035683:MicroRNAs; D006427:Hemin; D051547:Heme Oxygenase-1