(157bg) Regulation of Pancreatic Regenerating Protein (Reg) Expression By Microrna-7

Regenerating islet-derived (Reg) proteins display pro-proliferative, anti-apoptotic and differentiation-inducing actions in normal and disease states. However, little is known about the regulation of their expression. We tested our hypothesis that Reg expression is influenced by microRNAs. In silico analysis identified microRNA-7 (mir7) as a candidate targeting the murine Reg1 gene in pancreatic acinar and islet β-cells. The work opens avenues for discovering additional microRNAs influencing the expression of Reg members, thereby expanding our knowledge of the biology of Reg proteins and their role(s) in diseases such as diabetes and pancreatitis.

Rodent β-cell and acinar cell lines (ATCC, Manassas, VA) and HEK293 cells were cultured in DMEM medium with 10% fetal bovine serum (FBS) at 37 ^oC and 5% CO₂. Murine pancreatic islets and exocrine tissue were isolated. The levels of reg1 and mir7were probed by quantitative PCR (qPCR) or immunostaining. The 3’ untranslated region (3’UTR) of the Reg1 gene as well as the mir7 sequence were cloned in expression vectors upstream of the luciferase (Luc-3’UTR-reg1) and green fluorescent protein (GFP), respectively.

Lower expression of Reg1 was observed by immunostaining and qPCR in β-cells and primary islets compared to exocrine cells. This profile was inverted for the expression of mir7. Suppression of mir7 in beta-cells, which natively exhibit high mir7 levels, resulted in an increase in reg1. Overexpression of mir7 had the opposite effect on reg1. When the Luc-3’UTR-reg1 construct was delivered with a mir7 overexpression vector to exocrine cells, the luciferase activity was lower. This finding indicated that the microRNA directly interacts with the 3’UTR of reg1. Yet, mir7 did not affect the expression of the human orthologs of murine reg1, REG1A and REG1B. Current efforts focus on the identification of other microRNA moieties that directly target human Reg genes.

This is the first account of microRNA modulation of any Reg protein member directly, and our data warrant further studies. Given the link of Reg proteins to diabetes and various cancer types, including pancreatic ductal adenocarcinoma, the identification of microRNAs regulating Reg levels has the potential to contribute to the development of pertinent therapeutics and to extend our understanding of the pathophysiological role(s) of Reg.