Session: SUN 303-321-Cancer in Endocrine Tissues
Bench to Bedside
Poster Board SUN-316
Objective: To analyze the mRNA expression of Wnt/beta-catenin pathway genes (CTNNB1, AXIN1, AXIN2 and DKK3) in an adrenocortical cell line and to verify if the miR-449 hyperexpression could regulate the expression of these genes.
Patients and Methods: the effect of miRNA-449 hyperexpression was analyzed in the human adrenocortical carcinoma cell line NCI-H295A. The cells were concomitantly seeded in 6-well plates with 92-99% viability, transfected with miR-449 precursor at final concentration of 5 and 30nmol/l and the cells were incubated for 48h. All experiments were repeated 3 times, with five or more replicates in each independent experiment. We used a negative control in all experiments and miR-1 (target PTK9) as a positive control. Relative gene expression was measured by q PCR and calculated by 2-ΔΔCt method. The p.S45P CTNNB1mutation in H295A was investigated by automatic sequencing. Statistics: one way ANOVA and Mann-Whitney test; P<0.05. MiR-449 targets were analyzed by miRMAP.
Results: NCI-H295A and NCI-H295R cell lines exhibited different genotype regarding CTNNB1 mutation status. The p.S45P CTNNB1 mutation was previously found in heterozygosis. However, we found this mutation in homozygosis in NCI-H295A cell line. MiRMAP analysis identified AXIN1 and AXIN2 as miR-449 targets. MiR-449 mRNA expression was higher in transfected than in untransfected H295A cells (12 to 49 x 103fold; p<0.0001). In basal conditions NCI-H295A cells express CTNNB1, AXIN1, AXIN2 and DKK3 mRNA, however the hyperexpression of miR-449 did not modify the expression of these genes.
Conclusion: Although in silico analysis predicted AXIN1 and AXIN2 as potential miR-449 targets, our in vitro data did not confirm this prediction. Therefore, although hyperexpressed in ACTs, miR-449 does not appear to be involved in the Wnt/beta-catenin dysregulation found in these tumors.
Nothing to Disclose: LFL, LMC, DCG, FBC, MD, SRA
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