miR-449 does not regulate Wnt pathway genes in adrenocortical tumor cell line

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 303-321-Cancer in Endocrine Tissues
Bench to Bedside
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-316
Leticia Ferro Leal*, Leandro Machado Colli, Debora Cristiane Gomes, Fernanda Borchers Coeli-Lacchini, Margaret De Castro and Sonir Roberto Antonini
School of Medicine of Ribeirao Preto-University of Sao Paulo, Ribeirao Preto-SP, Brazil
Introduction: CTNNB1 mutations and increased beta-catenin expression and/or Wnt/beta-catenin pathway activation are frequent in ACTs. Of notice, AXIN1 differential expression and AXIN2 mutations were found in childhood and adult ACTs, respectively (1, 2). We previously observed higher miR-449 expression in childhood ACTs. A functional link between miRNA-449 and the Wnt signaling pathway was previously shown in PPNAD (3).

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 CTNNB1AXIN1AXIN2 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.

(1)Leal LF et al. J Clin Endocrinol Metab. 2011 Oct; 96(10):3106–3114. (2)Chapman A et al. J Clin Endocrinol Metab. 2011 Sep; 96(9):E1477-81(3)Iliopoulos D et al. Cancer Res. 2009 Apr; 69(8):3278-82.

Nothing to Disclose: LFL, LMC, DCG, FBC, MD, SRA

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: FAPESP and Cnpq