SUN-718 Reciprocal Regulation of miR-375 and ICER in Pancreatic Beta Cells

Abstract

MicroRNA-375 (miR-375) is overexpressed in people with type 2 diabetes (T2D) and has been linked to decreased insulin secretion and beta cell proliferation. Investigation into the transcription factor inducible cAMP early repressor (ICER) as an intermediate regulator of miR-375 was proposed because both are regulated by the cAMP pathway. This overexpression of miR-375 in T2D led us to hypothesize that beta cells with elevated and reduced levels of miR-375 will result in decreased and increased glucose-stimulated insulin secretion (GSIS), respectively. Results showed that when miR-375 was overexpressed, GSIS decreased by 61% when compared to a control in 25 mM glucose. Results showed that when miR-375 was inhibited, GSIS increased 6% when compared to a control in 25 mM glucose. In human islets, we found that inhibiting miR-375 led to an average 19% increase in GSIS, though due to the variability of human tissue these data were not significant (N=5). To investigate ICER’s binding affinity to the miR-375 promoter, a luciferase reporter assay was conducted. HEK-293 (human embryonic kidney) cells that were transfected with a luciferase reporter plasmid containing a cAMP recognition element (CRE) and a plasmid driving the overexpression of ICER had a 75% decrease when compared to our control (P<0.05). When transiently-expressed ICER was knocked down via siRNA, promoter activity increased by 13.1-fold (P< 0.05). Using a chromatin immunoprecipitation assay we found that an ICER antibody pulled down the rat miR-375 promoter an average of 13-fold compared with a control antibody (N=2). Additionally, because of a sequence alignment showing possible binding of miR-375 to the human ICER transcript we hypothesize that the two are in a negative feedback loop and can regulate each other’s expression. To investigate the double negative feedback loop a plasmid was constructed containing the GFP reporter gene and either the human or rodent ICER 3’UTR predicted miR-375 binding site. The GFP reporter assay was conducted to determine if miR-375 binds to ICER’s microRNA recognition element (MRE) in a species-specific way. In our GFP reporter experiment, data shows there is a 50% reporter gene decrease between our negative control and the human ICER MRE (N=4, P=0.017). Understanding microRNA gene regulation in the pancreas may have important implications for patients with T2D. MiR-375 may have the ability to interact with human ICER in a double negative feedback loop in the cAMP second messenger pathway, which will further clarify cellular mechanisms to potentially improve T2D drugs.