Abstract
miRNAs are predicted to control the activity of approximately 60% of all protein-coding genes participating in the regulation of several cellular processes and diseases, including cancer. Recently, we have demonstrated that miR-187 is significantly downregulated in prostate cancer (PCa) and here we propose a proteomic approach to identify its potential targets. For this purpose, PC-3 cells were transiently transfected with miR-187 precursor and miRNA mimic negative control. Proteins were analyzed by a two-dimensional difference gel electrophoresis (2D-DIGE) and defined as differentially regulated if the observed fold change was ±1.06. Then, MALDI-TOF MS analysis was performed after protein digestion and low abundance proteins were identified by LC–MS/MS. Peptides were identified by searching against the Expasy SWISS PROT database, and target validation was performed both in vitro by western blot and qRT-PCR and in clinical samples by qRT-PCR, immunohistochemistry and ELISA. DIGE analysis showed 9 differentially expressed spots (p<0.05) and 7 showed a down-regulated expression upon miR-187 re-introduction. Among these targets we identified aldehyde dehydrogenase 1A3 (ALDH1A3). ALDH1A3 expression was significantly downregulated in PC3, LNCaP and DU-145 cells after miR-187 re-introduction. Supporting these data, the expression of ALDH1A3 was found significantly (p<0.0001) up-regulated in PCa samples and inversely correlated (p<0.0001) with miR-187 expression, its expression being directly associated with Gleason score (p = 0.05). The expression of ALDH1A3 was measured in urine samples to evaluate the predictive capability of this biomarker for the presence of PCa and, at a signification level of 10%, PSA and also ALDH1A3 were significantly associated with a positive biopsy of PCa. In conclusion, our data illustrate for the first time the role of ALDH1A3 as a miR-187 target in PCa and provide insights in the utility of using this protein as a new biomarker for PCa.
Highlights
Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer death in men [1]
By base pairing to mRNAs, microRNAs mediate translational repression or mRNA degradation [4]
Previous results obtained by Yang et al [6] and Schramedei et al [15] confirmed that less than 10% of proteins identified by a proteomic approach were predicted by commonly used algorithms such as Pictar, Targetscan and miRanda
Summary
Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer death in men [1]. Several studies suggest that an individual miRNA can regulate hundreds of targets [6] and can function either as a tumor suppressor or oncogene, depending on the target genes [7], as well as contributing to the initiation and development of various types of cancer, including PCa [5]. Using miRNA microarray analysis (NCBI Gene Expression Omnibus database accession number GSE45604), our group identified miR-187 as a tumor suppressor miRNA in PCa [8]. This study used a proteomic approach based on two-dimensional gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis and, for the first time, identified ALDH1A3 as a miR187 target in PCa. In addition, the potential utility of ALDH1A3 as a tumor biomarker was evaluated
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