Masters Thesis

cAMP dependent regulation of miR-375 by ICER

The area of study for this project is a microRNA called miR-375 which 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 were found to be regulated by cAMP pathway. To investigate ICER’s binding affinity to the miR-375 promoter, a luciferase reporter assay was conducted. HEK-293T 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). Additionally, ICER’s expression was measured in human embryonic kidney cells (HEK-293T) when co-transfected with a plasmid containing ICER and a small interfering RNA (siRNA) using quantitative real time PCR (qPCR) and Western blot. A luciferase reporter assay showed a 13.1-fold increase from the miR-375 luciferase reporter plasmid containing the AP-1 promoter (p < 0.05). INS-1 cells that were transfected with a luciferase reporter plasmid the miR-375 promoter and a plasmid driving the overexpression of ICER had no significant fold change when compared to the control (p > 0.05). The goal of this project was to test the potential binding and regulation of the miR-375 promoter by ICER. The findings that I made indicate that there is potential binding and further investigation into this area could lead to a better understanding of the cAMP dependent regulation mechanism of miR-375.

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