Systematic Analysis of MicroRNAs Targeting the Androgen Receptor in Prostate Cancer Cells

Abstract

Androgen receptor (AR) is expressed in all stages of prostate cancer progression, including in castrationresistant tumors. Eliminating AR function continues to represent a focus of therapeutic investigation, but AR regulatory mechanisms remain poorly understood. To systematically characterize mechanisms involving microRNAs (miRNAs), we conducted a gain-of function screen of 1129 miRNA molecules in a panel of human prostate cancer cell lines and quantified changes in AR protein content using protein lysate microarrays. In this way, we defined 71 unique miRNAs that influenced the level of AR in human prostate cancer cells. RNA sequencing data revealed that the 3 0 UTR of AR (and other genes) is much longer than currently used in miRNA target prediction programs. Our own analyses predicted that most of the miRNA regulation of AR would target an extended 6 kb 3 0 UTR. 3 0 UTR-binding assays validated 13 miRNAs that are able to regulate this long AR 3'UTR

miR-145 suppress the androgen receptor in prostate cancer cells and correlates to prostate cancer prognosis

“…The observation that miR-145 is not regulating AR in LNCaP cells is supported by the LMA screening of miRNAs affecting AR in LNCaP cells where miR-145 is not identified (13). However, AR activity was found to be regulated by miR-145 in LNCaP cells, possibly indicating that miR-145 in addition to AR also regulates an AR cofactor or chaperon acting in synergy to influence the activity of AR.…”

“…In short, small RNA was extracted by a slight modified protocol of miRVana miRNA Isolation Kit (Life Technology) from formalin fixed paraffin embedded (FFPE) trans urethral of the prostate tissue (TURP) sections collected 1990 -1999 in Malmö, Sweden. AR immunohistochemistry has been performed on slides adjacent to these and total prostate specific antigen (PSA) was measured in serum from these patients at time of diagnosis (13). The study was approved by the Regional Ethical Review Board in Lund, all patients gave a written informed consent, and we have adhered to the Helsinki Declaration.…”

“…The AR protein content in the prostate tissues in our cohort has previously been determined by immunostaining and the staining scored by overall intensity (1; weak, 2; moderate to strong, 3; intense, Fig. 2C, lower panel) (13). When correlating miR-145 expression levels, measured by qRT-PCR on RNA extracted from an adjacent slide, with the semi-quantitative assessment of AR immunostaining in the malignant epithelial cells, a statistically significant inverse association was found (p<0.0001, Mann Whitney U test=0.0025, Student's t-test, Fig.…”

Cancer Biology, Toxicology and Alternative Methods Development Go Hand‐in‐Hand

“…The HTS experiments are mainly carried out to discover new and more selective cancer-associated pathways or processes [16,21]. To study pathway activation or repression caused by the drug candidates, reverse-phase protein lysate arrays (RPPA) or gene expression profiling are commonly employed thereafter [29,30]. Fig.…”

“…In stage 3, class-representative agents with high intrinsic potency for exerting toxic effects are typically subjected to even broader characterization taking into account the concentrations and time points defined in the stage 1 and 2 results. Additionally, if following stage 1 testing, only a low number of compounds has indicated toxicity, or if the toxicity mechanism remains unclear, one could conceptually see stages 2 and 3 being assayed simultaneously, or stage 2 could follow stage 3 to verify mechanisms inferred from genomics profiling [21,30]. Typically, the gene expression profiling analysis of stage 3 would serve to determine broad toxicology-relevant bioidentities of 'the selected few' group.…”