AbstractsBiology & Animal Science

The E3 Binding Protein of the Pyruvate Dehydrogenase Complex as a Possible Target of MicroRNA-29a

by Karen E. Witt

Institution: Roskilde University
Year: 2014
Record ID: 1122421
Full text PDF: http://rudar.ruc.dk/handle/1800/15300


Background: Diabetes is characterized by impaired glucose stimulated insulin secretion (GSIS) from the pancreatic b-cells. The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible decarboxylation of pyruvate to acetyl-CoA in the breakdown of glucose. The catabolism of glucose drives the formation of ATP, which triggers GSIS. It is shown that mir-29a is up-regulated in several tissues in response to hyperglycemia and that over-expression of miR-29a impairs GSIS, while depletion improves GSIS. Based on a target sequence analysis it was hypothesized that the 3’UTR of the E3 binding protein mRNA, a subunit of PDC, could be a potential target for binding of miR-29a. The expression of miR-29a would thus down-regulate the function of the entire complex. Methods: To investigate whether the 3’UTR of PDHx is a target of miR-29a a reporter gene analysis was conducted. A vector construct containing PDHx 3’UTR was transfected into rat insulinoma pancreatic β-cell lines (INS-1E), one of which was over-expressing miR-29a (miR-29a OE), while the other expressed a microRNA with no targets (NTC). The controls compared to PDHxUTR were miR-29a perfect, miR-29a mutated and Syntaxin-1a. A vector construct of a mutated PDHxUTR was designed in order to compare with the wild type PDHxUTR, the mutagenesis was however unsuccessful. The reporter vectors were co-transfected with a normalization vector expressing β-galactosidase or renilla luciferase. Additionally the reporter gene analysis was conducted in a transient miR-29a mimic system where INS-1E cells were transfected with the same set of vectors but with positive and negative mimics of miR-29a added to the cells instead of the constitutive endogenous expression. Results: An overlap-extension PCR and a whole plasmid PCR were employed in the mutagenesis of PDHxUTR. The mutagenesis was not successful, and the reporter gene analysis was therefore only performed for the wild type pMirTarget PDHxUTR. Transfections using the endogenous constitutive expression of miR-29a showed a significant decrease in normalizations factors β-galactosidase and RR luciferase activities between the NTC and miR-29a OE cell line for all reporter vectors. When lowering the concentration of normalization vector, the β-galactosidase activities became more similar between the cells. The protein concentrations differed significantly between the cell lines in the first transfections, indicating a different growth rate for the two cell lines. In the mimic system PDHxUTR showed a lower reporter gene activity when treated with positive mimic. Additionally, the controls yielded the expected results. Conclusion: Transfections using mimic oligos were superior to the endogenous constitutive transfections. The results indicate a binding of miR-29a to a target site in PDHxUTR. As the experiment was only performed once the results are non-conclusive, but it is advised that replicates are performed including the mutation of PDHxUTR to confirm the regulatory effect of miR-29a on PDHx.