title: MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells.
abstract: The role of beta cell microRNA (miR)-21 in the pathophysiology of type 1 diabetes has been controversial. Here, we sought to define the context of beta cell miR-21 upregulation in type 1 diabetes and the phenotype of beta cell miR-21 overexpression through target identification.
  
  
  
  Islets were isolated from NOD mice and mice treated with multiple low doses of streptozotocin, as a mouse model of diabetes. INS-1 832/13 beta cells and human islets were treated with IL-1β, IFN-γ and TNF-α to mimic the milieu of early type 1 diabetes. Cells and islets were transfected with miR-21 mimics or inhibitors. Luciferase assays and polyribosomal profiling (PRP) were performed to define miR-21-target interactions.
  
  
  
  Beta cell miR-21 was increased in in vivo models of type 1 diabetes and cytokine-treated cells/islets. miR-21 overexpression decreased cell count and viability, and increased cleaved caspase 3 levels, suggesting increased cell death. In silico prediction tools identified the antiapoptotic mRNA BCL2 as a conserved miR-21 target. Consistent with this, miR-21 overexpression decreased BCL2 transcript and B cell lymphoma 2 (BCL2) protein production, while miR-21 inhibition increased BCL2 protein levels and reduced cleaved caspase 3 levels after cytokine treatment. miR-21-mediated cell death was abrogated in 828/33 cells, which constitutively overexpress Bcl2. Luciferase assays suggested a direct interaction between miR-21 and the BCL2 3' untranslated region. With miR-21 overexpression, PRP revealed a shift of the Bcl2 message towards monosome-associated fractions, indicating inhibition of Bcl2 translation. Finally, overexpression in dispersed human islets confirmed a reduction in BCL2 transcripts and increased cleaved caspase 3 production.
  
  
  
  In contrast to the pro-survival role reported in other systems, our results demonstrate that miR-21 increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation.
authors.affiliation: Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Drive, MS2031, Indianapolis, IN, 46202, USA. eksims@iu.edu.|Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA. eksims@iu.edu.|Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA. eksims@iu.edu.
authors.forename: Emily K
authors.initials: EK
authors.lastname: Sims
authors.affiliation: Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Drive, MS2031, Indianapolis, IN, 46202, USA.|Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA.|Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Lakhter
authors.affiliation: Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Drive, MS2031, Indianapolis, IN, 46202, USA.|Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Anderson-Baucum
authors.affiliation: Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Drive, MS2031, Indianapolis, IN, 46202, USA.|Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.|Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Tatsuyoshi
authors.initials: T
authors.lastname: Kono
authors.affiliation: Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Drive, MS2031, Indianapolis, IN, 46202, USA.|Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Tong
authors.affiliation: Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Drive, MS2031, Indianapolis, IN, 46202, USA.|Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.|Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.|Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA.|Richard L. Roudebush VA Medical Center, Indianapolis, IN, USA.
authors.forename: Carmella
authors.initials: C
authors.lastname: Evans-Molina
mesh_terms: D000818:Animals; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D050417:Insulin-Secreting Cells / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D053583:Interleukin-1beta; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; C546082:mirn21 microRNA, rat; D007371:Interferon-gamma
keywords: Animal – mouse; Basic science; Beta cell signal transduction; Cell lines; Islet degeneration and damage; Islets