title: MicroRNA-146 inhibits thrombin-induced NF-κB activation and subsequent inflammatory responses in human retinal endothelial cells.
abstract: OBJECTIVE
  Nuclear factor-κB (NF-κB), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-κB through protease-activated receptor (PAR)-1, a member of the G-protein-coupled receptor (GPCR) superfamily, and contributes to DR. The current study is to uncover the roles of microRNA (miRNA) in thrombin-induced NF-κB activation and retinal endothelial functions.
  
  
  METHODS
  Target prediction was performed using the TargetScan algorithm. Predicted target was experimentally validated by luciferase reporter assays. Human retinal endothelial cells (HRECs) were transfected with miRNA mimics or antimiRs and treated with thrombin. Expression levels of miR-146 and related protein-coding genes were analyzed by quantitative (q)RT-PCR. Functional changes of HRECs were analyzed by leukocyte adhesion assays.
  
  
  RESULTS
  We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-κB activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-κB-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-κB activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-κB through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs.
  
  
  CONCLUSIONS
  We uncovered a novel negative feedback regulatory mechanism on thrombin-induced GPCR-mediated NF-κB activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-κB activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-κB activation pathways and related inflammatory processes in DR.
authors.affiliation: Department of Pharmacology, Rush University Medical Center, Chicago, Illinois, United States.
authors.forename: Colleen
authors.initials: C
authors.lastname: Cowan
authors.affiliation: Department of Ophthalmology, Kresge Eye Institute, School of Medicine, Wayne State University, Detroit, Michigan, United States Department of Anatomy & Cell Biology, School of Medicine, Wayne State University, Detroit, Michigan, United States.
authors.forename: Chithra K
authors.initials: CK
authors.lastname: Muraleedharan
authors.affiliation: Department of Pharmacology, Rush University Medical Center, Chicago, Illinois, United States.
authors.forename: James J
authors.initials: JJ
authors.lastname: O'Donnell
authors.affiliation: Department of Anatomy & Cell Biology, School of Medicine, Wayne State University, Detroit, Michigan, United States.
authors.forename: Pawan K
authors.initials: PK
authors.lastname: Singh
authors.affiliation: Department of Pharmacology, Rush University Medical Center, Chicago, Illinois, United States.
authors.forename: Hazel
authors.initials: H
authors.lastname: Lum
authors.affiliation: Department of Anatomy & Cell Biology, School of Medicine, Wayne State University, Detroit, Michigan, United States.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Pharmacology, Rush University Medical Center, Chicago, Illinois, United States.
authors.forename: Shunbin
authors.initials: S
authors.lastname: Xu
mesh_terms: D003930:Diabetic Retinopathy / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D035683:MicroRNAs / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism*; D012160:Retina / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology*; D015533:Transcriptional Activation
chemical_list: D006490:Hemostatics; C542542:MIRN146 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; D013917:Thrombin
keywords: NF-kB activation; diabetic retinopathy; microRNA; retinal endothelial cells; thrombin