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

• Published in: Diabetologia Vol. 60; no. 6; pp. 1057 - 1065
• Main Authors: Sims, Emily K., Lakhter, Alexander J., Anderson-Baucum, Emily, Kono, Tatsuyoshi, Tong, Xin, Evans-Molina, Carmella
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.06.2017; Springer Nature B.V
• Subjects: 3' Untranslated regions; Animal models; Animals; Apoptosis; Bcl-2 protein; Beta cells; Caspase; Caspase-3; Cell death; Cytokines; Diabetes; Diabetes mellitus; Diabetes Mellitus, Type 1 - genetics; Fluorescent Antibody Technique; Human Physiology; Humans; Immunoblotting; Insulin-Secreting Cells - metabolism; Interferon; Interferon-gamma - metabolism; Interleukin 1; Interleukin-1beta - metabolism; Internal Medicine; Lymphoma; Male; Medicine; Medicine & Public Health; Metabolic Diseases; Mice; Mice, Inbred NOD; MicroRNAs; MicroRNAs - genetics; MicroRNAs - metabolism; miRNA; Proto-Oncogene Proteins c-bcl-2 - genetics; Proto-Oncogene Proteins c-bcl-2 - metabolism; Rats; Rodents; Streptozocin; Transcription; Translation; Tumor Necrosis Factor-alpha - metabolism; Basic science; Beta cell signal transduction; Islets; Animal – mouse; Cell lines; Islet degeneration and damage
• ISSN: 0012-186X; 1432-0428
• DOI: 10.1007/s00125-017-4237-z

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.