Abstract
The Androgen Receptor (AR) plays a key role in prostate biology and in the progression of prostate cancer (PCa) to castration resistance. The role of microRNAs (miRNAs) in aberrant AR signaling have not been fully characterized. Here we screened a library of 810 miRNA mimics to identify miRNAs that alter AR activity in complementary functional assays including protein lysate microarray (LMA) quantification of AR and PSA protein levels, AR transcriptional reporter activity, and AR-positive PCa cell viability. Candidate AR-regulating miRNAs were verified through AR transcriptional reporter and cell viability assays. MiRNA binding sites were found within the AR 3′-untranslated region (UTR) and within the AR and AR-V7 coding regions. MiRNA activity was characterized by western blotting, 3′-UTR reporter assay, and AR-GFP and AR-V7-GFP reporter assays. Results uncovered miR-30 family members as direct AR inhibitors. Inhibition of endogenous miR-30b-3p and miR-30d-5p enhanced AR expression and androgen-independent cell growth. Droplet digital RT-PCR quantification of miR-30c-5p and miR-30d-5p revealed significantly reduced levels in metastatic castration resistant PCa (CRPC), when compared to healthy prostate tissues. MiR-30d-5p levels were inversely correlated with AR activity, as measured by PSA mRNA, in metastatic CRPC. Collectively, these studies provide a comprehensive evaluation of AR-regulating miRNAs in PCa.
Highlights
The Androgen Receptor (AR) is a ligand-activated nuclear receptor and transcription factor that is required for the natural development of the prostate [1]
There is little knowledge regarding the levels of AR-regulating miRNAs in the most relevant tissue, metastatic castration resistant PCa (CRPC), and correlative expression analyses between AR-targeting miRNAs and AR activity are lacking in these important tissues
A moderate, but statistically significant, correlation was observed between AR and PSA protein levels in LNCaP cells (Pearson r = 0.497, p < 0.001), VCaP cells (Pearson r = 0.301, p < 0.001), and LAPC4 cells (Pearson r = 0.536, p < 0.001) from this screen, suggesting that some of the PSA regulating mimics may act through AR suppression (Supplementary Figure S3)
Summary
The Androgen Receptor (AR) is a ligand-activated nuclear receptor and transcription factor that is required for the natural development of the prostate [1]. Most metastatic PCa cases develop resistance, often through the re-activation of AR signaling by AR gene mutation, amplification, over-expression, alternative splicing, post-translational modification, alteration of co-factor expression, or intratumoral androgen production [5,6,7]. There is little knowledge regarding the levels of AR-regulating miRNAs in the most relevant tissue, metastatic CRPC, and correlative expression analyses between AR-targeting miRNAs and AR activity are lacking in these important tissues. In light of this we sought to identify AR-regulating miRNAs, through a series of complementary functional screens, and to study their potential role in advanced disease through gene expression analyses in localized hormone sensitive PCa and metastatic CRPC
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