Identification of MicroRNA Expression in Gastrin Stimulated Adenocarcinoma Cells
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The Gastrin System Biology group studies the molecular responses to gastrin using cell line model systems. Recently microRNAs (miRNAs) have been acknowledged as essential participants in the regulation of gene expression, it is therefore of interest to identify the role of miRNAs in gastrin-mediated regulation. The aim of this master thesis project was to establish a method for miRNA identification in gastrin stimulated adenocarcinoma-cells, and to gain insight into regulation of specific miRNAs and their targets. A microRNA array profiling of RNA isolated from gastrin stimulated AR42J-cells were performed. Six miRNAs were found to be regulated as a result of gastrin stimulation, among these were miR-132 and miR-212. Subsequent qRT-PCR analyses confirm gastrin-induced up-regulation of miR-132 and miR-212. Our research group has found the transcription factor NR4A2 to be induced by gastrin both at the mRNA and protein level. Database searches using the TargetScan search-engine revealed NR4A2 as a predicted target for both miR-132 and miR-212. To determine whether miR-132 and miR-212 regulate NR4A2 expression, knock-down and knock-in of the microRNAs in gastrin treated AGS-2 cells were performed. The result shows that miR-212 most likely target NR4A2. To examine the biological role of miR-132 and miR-212 migration assays were performed with knock-in and knock-down of miR-132 or miR-212 in AGS-2 cells. Knock-in of miR-132/212 gave an increased migration, whereas knock-down of miR-132/212 gave both increased and decreased migration. The results indicate that both microRNAs may act as tumor suppressor- and onco-miRNAs. CREB, CREM/ICER, CBP and TORC2 are found to be involved in the gastrin-mediated signaling, and it was therefore of interest to determine whether NR4A2 interact with some of these proteins. However, co-immunoprecipitation experiments did not show any interaction between NR4A2 and theses proteins.