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

Research output: Contribution to journalArticle

22 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalDiabetologia
DOIs
StateAccepted/In press - Mar 9 2017

Fingerprint

B-Cell Lymphoma
MicroRNAs
Apoptosis
Messenger RNA
Type 1 Diabetes Mellitus
Islets of Langerhans
Caspase 3
Cell Death
Luciferases
Cytokines
Inbred NOD Mouse
3' Untranslated Regions
Streptozocin
Interleukin-1
Computer Simulation
Cell Survival
Proteins
Up-Regulation
Cell Count

Keywords

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

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells. / Sims, Emily K.; Lakhter, Alexander J.; Anderson-Baucum, Emily; Kono, Tatsuyoshi; Tong, Xin; Evans-Molina, Carmella.

In: Diabetologia, 09.03.2017, p. 1-9.

Research output: Contribution to journalArticle

Sims, Emily K. ; Lakhter, Alexander J. ; Anderson-Baucum, Emily ; Kono, Tatsuyoshi ; Tong, Xin ; Evans-Molina, Carmella. / MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells. In: Diabetologia. 2017 ; pp. 1-9.
@article{e158e1d7b5d740648fa3b0a9da217d0b,
title = "MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells",
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",
author = "Sims, {Emily K.} and Lakhter, {Alexander J.} and Emily Anderson-Baucum and Tatsuyoshi Kono and Xin Tong and Carmella Evans-Molina",
year = "2017",
month = "3",
day = "9",
doi = "10.1007/s00125-017-4237-z",
language = "English (US)",
pages = "1--9",
journal = "Diabetologia",
issn = "0012-186X",
publisher = "Springer Verlag",

}

TY - JOUR

T1 - MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells

AU - Sims, Emily K.

AU - Lakhter, Alexander J.

AU - Anderson-Baucum, Emily

AU - Kono, Tatsuyoshi

AU - Tong, Xin

AU - Evans-Molina, Carmella

PY - 2017/3/9

Y1 - 2017/3/9

N2 - 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.

AB - 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.

KW - Animal – mouse

KW - Basic science

KW - Beta cell signal transduction

KW - Cell lines

KW - Islet degeneration and damage

KW - Islets

UR - http://www.scopus.com/inward/record.url?scp=85014685856&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85014685856&partnerID=8YFLogxK

U2 - 10.1007/s00125-017-4237-z

DO - 10.1007/s00125-017-4237-z

M3 - Article

C2 - 28280903

AN - SCOPUS:85014685856

SP - 1

EP - 9

JO - Diabetologia

JF - Diabetologia

SN - 0012-186X

ER -