Diabetologia

, Volume 60, Issue 6, pp 1057–1065 | Cite as

MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells

  • Emily K. Sims
  • Alexander J. Lakhter
  • Emily Anderson-Baucum
  • Tatsuyoshi Kono
  • Xin Tong
  • Carmella Evans-Molina
Article

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.

Keywords

Animal – mouse Basic science Beta cell signal transduction Cell lines Islet degeneration and damage Islets 

Abbreviations

AOPI

Acridium orange/propidium iodide

Atf4

Activating transcription factor 4

BCL2

B cell lymphoma 2

BiP

Binding immunoglobulin protein

Chop

C/EBP homologous protein

ER

Endoplasmic reticulum

GSIS

Glucose-stimulated insulin secretion

LNA

Locked nucleic acid

miR-21

miRNA 21

miRNA

microRNA

MLD-STZ

Multiple low-dose streptozotocin

PDCD4

Programmed cell death 4

PRP

Polyribosomal profiling

qPCR

Quantitative real-time PCR

ROS

Reactive oxygen species

STZ

Streptozotocin

UTR

Untranslated region

Supplementary material

125_2017_4237_MOESM1_ESM.pdf (730 kb)
ESM(PDF 729 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Emily K. Sims
    • 1
    • 2
    • 3
  • Alexander J. Lakhter
    • 1
    • 2
    • 3
  • Emily Anderson-Baucum
    • 1
    • 4
  • Tatsuyoshi Kono
    • 1
    • 3
    • 4
  • Xin Tong
    • 1
    • 4
  • Carmella Evans-Molina
    • 1
    • 3
    • 4
    • 5
    • 6
  1. 1.Center for Diabetes and Metabolic DiseasesIndiana University School of MedicineIndianapolisUSA
  2. 2.Department of PediatricsIndiana University School of MedicineIndianapolisUSA
  3. 3.Herman B. Wells Center for Pediatric ResearchIndiana University School of MedicineIndianapolisUSA
  4. 4.Department of MedicineIndiana University School of MedicineIndianapolisUSA
  5. 5.Department of Biochemistry and Molecular BiologyIndiana University School of MedicineIndianapolisUSA
  6. 6.Richard L. Roudebush VA Medical CenterIndianapolisUSA

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