MiR‐21 Contributes to Cytokine‐Induced Beta Cell Dysfunction via Inhibition of mRNAs Regulating Beta Cell Identity
Authors: Ibrahim Sara Mohommad , Mirmira Raghavendra , Anderson Ryan , Sims Emily 
Published: June 2021

A hallmark of diabetes is the loss of physical or functional β cell mass. Alterations in β cell microRNA (miRNA) profiles have been described in diabetes. MiRNAs have also been shown to serve as important regulators of β cell development and function, implicating them in β cell dysfunction during diabetes development. Our lab has previously demonstrated that β cell microRNA 21 (miR‐21) is increased in models of diabetes. However, a comprehensive analysis of the β cell effects of miR‐21 remain poorly defined, and the effects of miR‐21 on in vivo glucose homeostasis have never been explored. To this end, we performed a comprehensive in silico analysis of bioinformatics databases to identify potential β cell targets of miR‐21, which yielded multiple targets in the Transforming Growth Factor Beta 2 (Tgfb2) and Fibroblast Growth Factor Receptor 3 (Fgfr3) pathways associated with regulation of differentiation. We hypothesize that β cell miR‐21 plays a critical role in inhibiting β cell function and inducing loss of β cell identity. To validate targets in vitro, we developed a model whereby miR‐21 is upregulated using a dose dependent lentiviral Tetracycline‐on system in INS1 cells. Overexpression of miR‐21 led to a reduction in expression levels of several members of the Tgfb2 and Fgfr3 pathways as well as multiple transcription factors associated with β cell function and identity, and an increase in aldehyde dehydrogenase transcripts, consistent with β cell dedifferentiation. To verify direct interactions between miR‐21 and candidate target mRNAs, a biotin pulldown experiment was performed using a 3′ biotinylated mature miR‐21 construct and a 3′ biotinylated cel‐miR‐67 control construct. Several mRNAs associated with β cell identity were enriched in the pulldown, indicating a direct interaction with miR‐21. Lineage tracing was performed within an in vivo zebrafish model of β cell specific oxidative stress in which β cells expressed a nuclear GFP signal. Whole body knock down of miR‐21 by morpholino microinjection showed a protective effect in stressed β cells and rescued against a dedifferentiated phenotype. To test the effect of miR‐21 on glucose tolerance in vivo, inducible β cell specific knockout (βmiR‐21KO) and overexpression (βmiR‐21) mice were generated by crossing Ins1tm1(CreERT2)Thor mice with miR‐21 floxed mice and miR‐21‐CAG‐Z‐EGFP mice, respectively. When compared to littermate controls, intraperitoneal glucose tolerance tests (IPGTT) exhibited hyperglycemia in βmiR‐21 mice and euglycemia in βmiR‐21KO mice. Metabolic studies, including glucose stimulated insulin secretion (GSIS) and insulin tolerance tests (ITT) are ongoing in our mouse models. Our results implicate miR‐21 as a regulator of β cell dedifferentiation during diabetes development.Support or Funding InformationDeVault Fellowship through the Indiana University School of MedicineThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.