Genome-wide functional analysis of microRNAs in prostate cancer

MicroRNAs (miRNAs) are a novel class of negative gene regulators that have a major modulatory impact on the protein expression levels. They base-pair primarily to the 3'-untranslated region (3'UTR) of their target mRNAs leading to translational repression or transcript degradation. Thus far, studies on differential expression of miRNAs in prostate cancer have been published but functional proof of their impact in prostate cancer pathogenesis remains largely unexplored. In this study, we aimed to systematically identify cancer-associated miRNAs and their functional significance by combining data from overexpression phenotypes of individual miRNAs, with expression profiles of miRNA and mRNA. We screened two large-scale miRNA libraries one from Ambion (pre-miR V2; with 319 molecules) and one from Dharmacon (Mimic v.10.1; 820 molecules). The miRNAs were reverse-transfected in 384-well plates that were incubated for 72 h. Cell proliferation was measured by CellTiter-Glor, and apoptosis by Apo-ONEr (Promega). The screens were done in two biological replicates with several malignant and non-malignant prostate cell lines (LNCaP, LAPC4, MDA-PCa-2b, 22Rv1, CWR-1R, RWPE-1, and EP156T). In the secondary validation and follow-up screens, further phenotypic analyses were performed by utilizing the protein lysate microarray (LMA) technology with antibodies against markers for proliferation (Ki67) and apoptosis (cleaved PARP). Anti-proliferative effects of mir-15a and mir-16 were evident in LNCaP and LAPC4 cells both in the primary screens and in LMA. In addition to these miRNAs, with previously described links to prostate cancer, several novel ones were identified. However, detailed molecular mechanisms and the signalling pathways targeted by these miRNAs require further exploration. For example, we are combining predicted protein target data from these miRNAs with simultaneously measured expression profiles from mRNAs and miRNAs in order to narrow the search to the most important target proteins. In summary, we are combining various functional methods to identify miRNA genes and their mechanisms of action on prostate cancer development and progression