A Role for MicroRNA-146a-5p Mediated Regulation of Stromal Interaction Molecule 1 and Store Operated Calcium Entry in the Pancreatic Beta-Cell in Response to Cytokine Mediated Stress

Abstract

Store-operated Ca2+ entry (SOCE) is involved in the maintenance of endoplasmic reticulum (ER) Ca2+ levels. The SOCE involves Stromal Interaction Molecule 1 (STIM1), distributed throughout the ER, and Orai1 channels, dispersed on the plasma membrane. SOCE is activated by the depletion of ER Ca2+ causing STIM1 to induce ER expansion and recruits Orai1 channels thus replenishing ER Ca2+. We reported downregulation of STIM1 in human islets from donors with type 2 diabetes (T2D) and in INS-1 β-cells treated with cytokines, and loss of STIM1 expression impairs β-cell SOCE, ER stress, and reduced insulin secretion. However, the regulatory mechanisms of STIM1 downregulation are unknown. To test this, actinomycin D and cycloheximide chase assay was performed to define whether IL-1β treatment impacted STIM1 mRNA or protein half-life. IL-1β had no impact on mRNA or protein decay. MicroRNAs (miRNAs), a class of small non-coding RNAs can regulate gene expression post-transcriptionally by binding to complementary regions in the 3’ untranslated region (UTR) of target mRNAs, affecting mRNA stability and translatability. The objective of this study was to establish miRNA regulation of STIM1 expression and altered SOCE. To identify potential miRNA candidates, RNA sequencing was done in human islets, treated with IL-1β and IFN-γ for 24 hrs. A total of 20 miRNAs were differentially expressed using a FC value of ≥ 1.5 and a p value of < 0.05. Of these, two miRNAs (miR-146a-5p and miR-4640-5p) were predicted by TargetScan to bind the 3’UTR of STIM1.To validate these findings, INS-1 β-cells, and human islets were treated with or without IL-1β. Only miR-146a-5p was upregulated in both systems. Consistent with inverse correlation, INS-1 β-cells transfected with miR-146a-5p mimic showed reduced STIM1 expression. To test whether miR-146a-5p inhibition preserves STIM1 expression, INS1 cells were treated with miR-146a-5p inhibitor along with IL-1β and inhibition of miR-146a-5p led to partial preservation of STIM1 expression. Future studies will test the effect of miR-146a-5p mimics and inhibitors on SOCE. The results indicate that the stress induced by IL-1β leads to induction of miR-146a-5p, which may then target STIM1 mRNA. Such studies could enable broader implementation of miRNA in βcell dysfunction.