MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells
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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.
KeywordsAnimal – mouse Basic science Beta cell signal transduction Cell lines Islet degeneration and damage Islets
Acridium orange/propidium iodide
Activating transcription factor 4
B cell lymphoma 2
Binding immunoglobulin protein
C/EBP homologous protein
Glucose-stimulated insulin secretion
Locked nucleic acid
Multiple low-dose streptozotocin
Programmed cell death 4
Quantitative real-time PCR
Reactive oxygen species
We acknowledge P. Fueger of Indiana University (Department of Pediatrics) and C. Newgard of Duke University, NC, USA (Departments of Medicine and Pharmacology and Cancer Biology) for their generous donation of INS-1 828/33 cells, and the Indiana University Center for Diabetes and Metabolic Diseases’ Islet and Physiology Core for assistance with islet isolation. We also thank Y. Gu (Pediatrics, Indiana University) for technical assistance. Some of the data included in this manuscript were previously presented in abstract form at the Midwest Islet Club, the Combined Annual Meeting of the American Federation for Medical Research/Central Society for Clinical and Translational Research, the Pediatric Endocrine Society meeting, and the 76th Scientific Sessions of the American Diabetes Association, in 2016.
The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.
This manuscript was also supported by funding from NIDDK K08DK103983 to EKS, a Pediatric Endocrine Society Clinical Scholar Award to EKS, a pilot and feasibility award within the Center for Diabetes and Metabolic Diseases NIH/NIDDK Grant Number P30 DK097512, funding by Indiana University Health and the Indiana Clinical and Translational Sciences Institute to EKS, funded in part by Grant no. KL2TR001106 and Grant no. UL1TR001108, NIH Grant no. 32DK064466 to AJL, 1F32DK104501-01A1 to EA-B, and NIH grant R01 DK093954, VA Merit Award I01BX001733 and JDRF SRA-2014-41-Q-R (to CEM). This study used core services provided by the Diabetes Research Center grant P30 DK097512 to Indiana University School of Medicine.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
EKS conceived and designed the experiments, acquired and analysed the data, and drafted and revised the manuscript. AJL acquired and analysed the data and revised the manuscript. EAB conceived the experiments, acquired and analysed the data, and revised the manuscript. TK conceived and designed the experiments, and revised the manuscript. XT contributed to concept and design, acquired and analysed the data, and revised the manuscript. CEM contributed to conception and design, interpreted/analysed the data, and drafted and revised the manuscript. All authors gave final approval of the version to be published. EKS is responsible for the integrity of the work as a whole.
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