Fig 3. Validation of ALDH1A3 as target of miR-187.

A) RNA22 predicted mRNA-miRNA heteroduplexes. RNA22 v1.0 software predicted three different miR-187 binding sites within ALDH1A3 mRNA sequence. Putative miR-187 binding sites were found in ALDH1A3 5’UTR region (i), coding region (CDS) (ii and iii) and 3’UTR (iv). All of them accomplish the criteria of a base pair minimum value of 14 and a maximum folding energy of -25. B) Western Blot analysis shows a reduction in the expression of the protein ALDH1A3 upon the re-introduction of miR-187 (miR-187 miRNA mimic transfected cells), confirming the inhibitory effect of the miRNA through its putative target. Cells were transfected with miR-187 miRNA mimic or negative control and harvested after 72h. Total cell extracts were prepared and electrophoresed by SDS-PAGE, followed by immunoblotting with anti-ALDH1A3 antibody. To ensure equal protein loading the membrane was immunoblotted with anti β-actin antibody. C) Luciferase reporter assay performed with firefly luciferase under control of the ALDH1A3 3′ UTR confirm the inhibition of ALDH1A3 mRNA expression (20% decrease of luciferase signal) upon miR-187 re-introduction. Data are presented relative to the vector control assigned a value of 100. The mean ± s.e.m. of three independent experiments is shown. D) Overexpression of ALDH1A3 mRNA was confirmed in a cohort of human prostate tumors (n = 96). There was an inverse correlation (not statistically significant) between the down-regulation of miR-187 found in these samples and the up-regulation of ALDH1A3.