ID: 84: β CELL DERIVED MIR-21 INCREASES APOPTOSIS VIA TRANSLATIONAL INHIBITION OF THE ANTIAPOPTOTIC PROTEIN BCL2 AND COULD SERVE AS A BIOMARKER OF TYPE 1 DIABETES MELLITUS

BackgroundAltered β cell and serum microRNA (miRNA) profiles have been described in Type 1 Diabetes (T1D), suggesting a role for these small, non-coding RNAs in β cell pathophysiology. Whereas previous reports have demonstrated increased expression of β cell miR-21 in models of T1D, the role of miR-21 in the β cell under pro-inflammatory conditions has been controversial.ObjectivesOur goal was to define the effect of miR-21 on β cell survival, through identification of miR-21 regulated gene targets. Furthermore, we sought to identify whether circulating extracellular vesicle (EV) β cell-derived miR-21 may reflect inflammatory stress within the islet during T1D development.Design/MethodsINS-1 828/13 or 828/33 β cells were treated with a cytokine mix to mimic the early T1D milieu. Islets were isolated from NOD mice with autoimmune diabetes and mice treated with multiple low dose streptozotocin (STZ) to induce chemical diabetes. To define the effects of miR-21 overexpression or inhibition, cells were transfected with a miR-21 mimic or inhibitor. In silico prediction tools were used to identify potential miR-21 targets. Luciferase assays and polyribosomal profiling (PRP) were performed to understand miR-21 and target interactions. EV RNA was isolated from media of cytokine treated cells and terminal serum of diabetic and normoglycemic control mice. Serum EV miR-21 concentration was assayed using digital droplet PCR.Resultsβ cell miR-21 was increased in cytokine treated INS-1 cells as well as islets from mouse models of T1D. miR-21 overexpression decreased β cell count and viability, and increased cleaved caspase 3 levels, suggesting increased apoptosis. In silico prediction tools identified the anti-apoptotic mRNA B Cell Lymphoma 2 (BCL2) as a conserved target of miR-21. Consistent with this, miR-21 overexpression decreased BCL2 protein expression. miR-21 inhibition had the opposite effect, increasing BCL2 protein levels and reducing cleaved caspase 3. Luciferase assay revealed decreased activation of the human BCL23' untranslated region (UTR) following miR-21 mimic treatment, suggesting a direct interaction between miR-21 and the BCL2 3'UTR. This effect was absent in cells transfected with a mutated BCL2 3'UTR. Preliminary polyribosomal profiling of cells overexpressing miR-21 revealed a shift of BCL-2 message toward monosomal fractions, indicating inhibition of BCL2 translation. The proapoptotic effect of overexpression was abrogated in 928/33 cells, which constitutively overexpress BCL-2. Cytokine treatment increased miR-21 levels in treated cell media 5-fold. This effect was doubled in EVs isolated from media. Although no increase in serum EVs existed in mice with chemical diabetes induced by STZ, when compared to controls, serum EV miR-21 was more than doubled in NOD mice with autoimmune diabetes.ConclusionsIn contrast to a prosurvival role in other systems, our results demonstrate that miR-21 increases β cell apoptosis, in part, via binding of the BCL2 mRNA 3'UTR to inhibit translation. In addition, our results suggest that cytokine-induced miR-21 production in β cells could increase circulating miR-21 levels during T1D development. Future work will identify new targets of β cell miR-21 and examine its role in vivo using transgenic mouse models, as well as verify elevations of exosomal miR-21 in serum samples from prediabetic mice and humans with developing T1D.