Non-coding RNAs comprise of several classes of RNA with diverse functions and include microRNAs and also RNAs encoded by ultra-conserved genomic regions. MicroRNAs target protein-coding genes and regulate their expression, usually by inducing mRNA degradation and/or by inhibiting the translation of mRNA into protein. Numerous microRNAs have been shown to have oncogenic properties while others act like tumor suppressor genes. These microRNAs have been termed oncomiRs. An alteration in their expression is causatively linked to cancer development and can predict disease outcome. We had previously examined genome-wide expression of microRNAs and mRNAs in 60 primary human prostate tumors and 16 non-tumor prostate tissues in collaboration with Dr. Carlo Croce at Ohio State University. The analysis revealed that both key components of microRNA processing and numerous microRNAs were significantly altered in prostate tumors when compared with surrounding non-cancerous tissue. Tumor microRNAs were up- and down-regulated when compared with non-cancerous tissue and the expression profile of the tumors yielded a diagnostic microRNA signature. Notably, prostate tumors tended to express all members of the miR-106b-25 cluster at significantly higher levels than non-tumor prostate, which is consistent with the miR-106b-25 cluster having oncogenic properties in prostate tumor biology. The expression of miR-1 and miR-133 was consistently lower in tumors than in non-tumor prostate, indicating that these microRNAs may act as tumor suppressors. We are continuing this project by studying the function of the miR-1-133 and the miR-106b-25 clusters in prostate cancer. We generated lentiviral vectors that encode these microRNAs together with a reporter gene (eGFP or luciferase). Evaluation of the constructs in prostate cancer cells confirmed that infected cells overexpress the processed mature microRNAs and the reporter. These vectors are being used to study the function of the microRNA clusters in cancer cells in vitro and in vivo. Through cell sorting for reporter-positive cells, we isolated cell populations of PC3, LNCaP, DU145 cancer cells that overexpress the miR-1-133 cluster and populations of RWPE-1, 22Rv1 and DU145 cells that overexpress the miR-106b-25 cluster. These cells are either being inoculated into nude mice for xenograft growth or tail vein-injected for monitoring of tumor nodule formation in the lung as a surrogate for metastasis. Tumor growth and/or metastasis are compared with cells that also express the reporter gene but not a microRNA transgene. The phenotypic properties of these cells are also being examined in extracellular matrix protein, migration, and invasion assays in vitro. In addition, we began studying the specific functions of miR-1 and miR-106b. Increased expression of miR-1 in prostate cancer cell lines induced growth arrest, with many cells residing in S phase and not undergoing mitosis. Over-expression of miR-1 and miR-106b in LNCaP human prostate cancer cells generated distinct gene expression alterations with a significant down-regulation of predicted target genes for these microRNAs. The experiment indicated that miR-1 specifically targets key genes involved in the regulation of the actin microfilament network, but also genes involved in cell cycle, DNA replication and recombination. Other experiments revealed that a central executioner of apoptosis, caspase-7, is targeted and down-regulated by miR-106b. A connectivity analysis of the miR-1-induced gene expression profile suggested that expression of this microRNA leads to alterations similar to those in epigenetic drug-treated cells (5-azacytidine, decitabine, and two histone deacetylase (HDAC) inhibitors, vorinostat and valproic acid). Targeting of LNCaP cells with miR-1 siRNA led to up-regulation of several of the miR-1 target genes, revealing that endogenous miR-1 can suppress these targets although expression of miR-1 was found to be low and near the detection limit in these cells. To evaluate whether the down-regulated protein-coding genes are direct targets of miR-1 and miR-106b, we produced wild-type and mutant 3'UTR reporter constructs for candidate genes and could show that miR-1 directly targets fibronectin, LIM and SH3 protein 1 (LASP1), notch homolog 3 (notch3), prothymosin alpha, exportin-6, and macrophage differentiation-associated gene (MMD), among others, and that miR-106b directly targets caspase-7. Treatment of cancer cells with miR-106b siRNA led to increased expression of caspase-7. In another experiment, we treated prostate cancer cells with epigenetic drugs (5-azadeoxycytidine) and the histone deactylase inhibitor, trichostatin A, and observed that cells treated with a combination of both re-express the miR-1-133 cluster while the expression of the miR-106b-25 clusters was largely unaffected. Expression of miR-1-133 is generally low or undetectable in untreated human prostate cancer cells, indicating that the expression of miR-1-133 is silenced in these cells consistent with a tumor suppressor function of the microRNA cluster. Lastly, we started to perform fluorescence microscopy to localize miR-1 target genes and found changes in expression and the overall location of these proteins when miR-1 was over-expressed. Furthermore, these experiments showed that miR-1 alters F-actin staining patterns and inhibited filipodia formation by the cancer cells. We will continue to characterize the effects of miR-1 and miR-106b on cell cycle and apoptosis and will focus on the mechanisms by which miR-1 modulates the actin filament network and filipodia formation. In future research, we will test whether overexpression of selected miR target genes can reverse the microRNA-induced phenotype. In addition to studying the function of the miR-106b-25 cluster in human cell lines, we are generating mice with prostate-specific over-expression of the miR-106b-25 cluster. It is our hypothesis that constitutively elevated expression of microRNAs encoded by this cluster will lead to neoplastic alteration in the prostate. This project is at a point where we have screened male founders carrying germline expression cassettes for probasin promoter-driven expression of the cluster and of either a luciferase (pBSn-miR106 IRES Luc) or a luciferase eGFP fusion gene reporter (pBSn-miR106 ffLuc2 egfp). Screening identified potential founders with high, prostate-specific luciferase expression. We have previously characterized the expression profile of ultraconserved region-derived non-coding RNAs (ucRNAs) in prostate tumors and surrounding normal tissue using microarrays. We also found that some key ucRNAs are silenced by epigenetic mechanisms in human prostate cancer cells. Our lab is the first to explore genome-wide expression profiles of ucRNAs in prostate cancer and one of the first to explore regulatory mechanisms of these RNAs. However, quantification of ucRNAs by other methods than microarrays has been challenging. A number of problems make ucRNAs difficult to measure including their overall low expression in the tissues, small differential expression, and the tendency of ultraconserved regions to transcribe RNAs in sense and antisense direction, thereby interfering with the accuracy of PCR-based quantitative assays. Thus, we are exploring a new quantitative detection system called the NanoString nCounter Analysis System. The NanoString nCounter Analysis System for the direct quantification of UCR transcripts is a newly introduced technology that may rival quantitative real-time PCR and array based assays because of its ability to measure RNAs in a direct and absolute manner. No study to date has profiled ucRNAs in such detail, and these experiments, if successful, would allow for additional studies exploring ucRNAs in human biofluids and tissues for biomarker discovery.
