Emily K Sims1,2,3, Alexander J Lakhter4,5,6, Emily Anderson-Baucum4,7, Tatsuyoshi Kono4,6,7, Xin Tong4,7, Carmella Evans-Molina4,6,7,8,9. 1. Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Drive, MS2031, Indianapolis, IN, 46202, USA. eksims@iu.edu. 2. Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA. eksims@iu.edu. 3. Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA. eksims@iu.edu. 4. Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Drive, MS2031, Indianapolis, IN, 46202, USA. 5. Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA. 6. Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA. 7. Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA. 8. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA. 9. Richard L. Roudebush VA Medical Center, Indianapolis, IN, USA.
Abstract
AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/ INTERPRETATION: 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.
AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/ INTERPRETATION: 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.
Entities:
Keywords:
Animal – mouse; Basic science; Beta cell signal transduction; Cell lines; Islet degeneration and damage; Islets
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