期刊
DIABETOLOGIA
卷 60, 期 6, 页码 1057-1065出版社
SPRINGER
DOI: 10.1007/s00125-017-4237-z
关键词
Animal-mouse; Basic science; Beta cell signal transduction; Cell lines; Islet degeneration and damage; Islets
资金
- NIDDK [K08DK103983]
- Pediatric Endocrine Society Clinical Scholar Award
- Center for Diabetes and Metabolic Diseases NIH/NIDDK Grant [P30 DK097512]
- Indiana University Health
- Indiana Clinical and Translational Sciences Institute
- NIH [KL2TR001106, UL1TR001108, 32DK064466, 1F32DK104501-01A1, R01 DK093954]
- VA Merit Award [I01BX001733]
- JDRF [SRA-2014-41-Q-R]
- Diabetes Research Center [P30 DK097512]
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-1 beta, IFN-gamma and TNF-alpha 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.
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