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 language | English (US) |
|---|---|
| Pages (from-to) | 1-9 |
| Number of pages | 9 |
| Journal | Diabetologia |
| DOIs | |
| State | Accepted/In press - Mar 9 2017 |
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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 journal › Article
}
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 -