title: Differential regulation of microRNA-146a and microRNA-146b-5p in human retinal pigment epithelial cells by interleukin-1β, tumor necrosis factor-α, and interferon-γ.
abstract: OBJECTIVE
  The inflammatory response of the retinal pigment epithelium (RPE) is implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB pathway. The aim of the present study is to investigate the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines.
  
  
  METHODS
  Confluent cultures of RPE cells established from adult human donor eyes were treated with the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. The expression of microRNAs was analyzed by real-time PCR using total RNA fraction. The retinal pigment epithelial cell line ARPE-19 was employed to analyze the promoter activity of the genes encoding miR-146a and miR-146b-5p. STAT1-binding activity of oligonucleotides was analyzed by electrophoretic mobility shift assay. ARPE-19 cells were transiently transfected with miR-146a and miR-146b-5p mimics for the analysis of IRAK1 expression by western immunoblotting.
  
  
  RESULTS
  Real-time PCR analysis showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to proinflammatory cytokines (IFN-γ + TNF-α + IL-1β) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10, and IL-6, and a decrease in that for HMOX1. The miR-146a induction was more dependent on IL-1β, since its omission from the cytokine mix resulted in a greatly reduced response. Similarly, the induction of miR-146b-5p was more dependent on IFN-γ, since its omission from the cytokine mix minimized the effect. In addition, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of the JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with either miR-146a mimic or miR-146b-5p mimic.
  
  
  CONCLUSIONS
  Our results clearly show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is highly induced by proinflammatory cytokines (IFN-γ + TNF-α + IL-1β). The induction of miR-146a showed a dependency on IL-1β, while that of miR-146b-5p on IFN-γ. Our results show for the first time that miR-146b-5p expression is regulated by IFN-γ, potentially via the JAK/STAT pathway. These two microRNAs could play a role in inflammatory processes underlying age-related macular degeneration or other retinal degenerative diseases through their ability to negatively regulate the nuclear factor-κB pathway by targeting the expression of IRAK1.
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA. kuttyk@nei.nih.gov
authors.forename: R Krishnan
authors.initials: RK
authors.lastname: Kutty
authors.forename: Chandrasekharam N
authors.initials: CN
authors.lastname: Nagineni
authors.forename: William
authors.initials: W
authors.lastname: Samuel
authors.forename: Camasamudram
authors.initials: C
authors.lastname: Vijayasarathy
authors.forename: Cynthia
authors.initials: C
authors.lastname: Jaworski
authors.forename: Todd
authors.initials: T
authors.lastname: Duncan
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Cameron
authors.forename: Erik K
authors.initials: EK
authors.lastname: Flemington
authors.forename: John J
authors.initials: JJ
authors.lastname: Hooks
authors.forename: T Michael
authors.initials: TM
authors.lastname: Redmond
mesh_terms: D000328:Adult; D001483:Base Sequence; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology*; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology*; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D018836:Inflammation Mediators; D053583:Interleukin-1beta; C542542:MIRN146 microRNA, human; D035683:MicroRNAs; D050794:STAT1 Transcription Factor; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases