1241-P: Inhibition of MiR-146a-5p Prevents Mitochondrial Dysfunction and ß-Cell Apoptosis, and Improves Insulin Secretion

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that function as modulators of gene expression. We previously identified upregulation of miR-146a-5p in pancreatic islets treated with proinflammatory cytokines and in human pancreatic sections from individuals with type 1 diabetes. While other studies have associated overexpression of miR-146a-5p with β cell apoptosis and defective insulin secretion, the molecular mechanisms through which these effects are mediated remain elusive. To this end, we transduced MIN6 cells with lentiviral vectors (i) overexpressing and (ii) inhibiting the expression of miR-146a-5p and developed stable cell lines resistant to puromycin. Monoclonal cell populations were developed by single clone selection and then treated with/without proinflammatory cytokines (IL-1β, IFN-γ and TNF-α) for 24 hours. Further characterization of the cells was performed using mRNA sequencing, western blot, RT-PCR of cognate targets and Seahorse mitochondrial functional assays. Overexpression of miR-146a-5p led to increased expression of cleaved caspase-3 and reduced insulin secretion under basal and cytokine-treated conditions, whereas inhibition of miR-146a-5p reversed these effects. Similarly, miR-146a-5p inhibition increased mitochondrial copy number, mitochondrial membrane potential, basal respiration rate, maximal respiration rate, spare respiratory capacity, and ATP production, suggesting a protective effect on the mitochondria, even upon exposure to proinflammatory cytokines. Consistent with these findings, bioinformatic analysis of sequencing data and validation of targets such as MAPT, MYRIP, NRG1, GPX1 also showed enrichment of pathways related to insulin secretion, apoptosis, mitochondrial function. Overall, findings from our study show for the first time that miR-146a-5p upregulation may alter β cell health through reduced mitochondrial function. Disclosure P. Krishnan: None. F. Syed: None. S. A. Weaver: None. C. Lee: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc.