Non-coding RNAs comprise several classes of RNA with diverse functions and include microRNAs and RNAs encoded by ultraconserved genomic regions (ucRNA). MicroRNA expression is commonly altered in solid human tumors. Multiple microRNAs have been shown to have oncogenic properties, or 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. Recently, the Croce group at Ohio State University reported that expression of ucRNA is also altered in human cancer. ucRNAs are encoded by 481 ultraconserved regions that are defined as segments greater than 200 bp with perfect conservation among human, rat, and mouse genomes. In collaboration with Dr. Croce, we are investigating microRNA and ucRNA expression profiles of prostate tumors. We are also studying the function of selected microRNAs, e.g., miR-1, miR-106b-25 cluster, and the miR-200c-141 cluster, whose expression was found to be altered in human prostate tumors, using cell culture and mouse models. Initially, we had determined genome-wide expression of microRNAs and mRNAs in 60 primary prostate tumors and 16 non-tumor prostate tissues. The mRNA analysis revealed that key components of microRNA processing and several microRNA host genes, e.g., MCM7, were significantly up-regulated in prostate tumors. Consistent with these findings, tumors expressed the miR-106b-25 cluster, which maps to intron 13 of MCM7, at significantly higher levels than non-tumor prostate. The expression levels of other microRNAs, including the miR-1-133 cluster, miR-200c, and a number of miR-106b-25 cluster homologues, were also altered in prostate tumors. Additional differences in microRNA abundance were found between organ-confined tumors and those with extraprostatic disease extension, e.g., up-regulation of miR-200 functional homologues in tumors with extraprostatic extension. Lastly, we found evidence that some microRNAs are androgen-responsive and that tumor microRNAs influence transcript abundance of protein-coding target genes in the cancerous prostate. In summary, microRNA expression becomes altered with the development and progression of prostate cancer. Some of these microRNAs regulate the expression of cancer-related genes in prostate cancer cells and may alter the phenotype of these cells. In an ongoing study, we are analyzing the expression profiles for ucRNA in these samples. Preliminary results indicate that numerous ucRNAs are differentially expressed between tumor and surrounding normal tissue, by Gleason grade, and between organ-confined tumors and those with extraprostatic disease extension. We have attempted to validate the chip data by other methods, but so far had limited success. The problems are explained by the small differential expression of ucRNAs between tumor and normal, the overall low expression of many ucRNAs in the tissues, and the tendency of ucRNA loci to transcribe RNAs in sense and antisense direction, which can interfere with the accuracy of RT-based quantitative assays. In addition to our ucRNA validation work, we determined the ucRNA expression profile of LNCaP prostate cancer cells that were either untreated or treated with a synthetic androgen (R1881), the DNA hypomethylation agent, 5-azacytidine, or the histone deacetylase inhibitor, trichostatin A. These experiments revealed that several ucRNA loci are regulated by androgen, DNA methylation, and by chromatin silencing. We have continued our work to elucidate the function of miR-1 and the miR-106b-25 and miR-200c-141 clusters in prostate cancer. The miR-106b-25 and miR-200c-141 clusters will be evaluated in transgenic mice with a probasin-driven expression of these clusters in the mouse prostate. The targeting vectors have been constructed and are being evaluated in cell culture before they can be used to generate transgenic mice. Ultimately, we will cross the miR-106b-25 and miR-200c-141 transgenic mice into PTEN KO mice that are prone to develop prostate tumors. It is the aim if this experiment to examine how the two microRNA clusters modulate prostate cancer development in the PTEN KO mice. Two microRNAs are evaluated in cell culture using phenotypic assays. Mir-1 is being over-expressed in human prostate cancer cell lines that do not have any detectable levels of endogenous miR-1 expression. Up-regulation of miR-1 in these cells leads to growth arrest and alters the sensitivity of the cells to DNA damaging agents. The mechanism by which miR-1 causes these phenotypes is currently studied. The function of miR-106b is also studied in cell culture models. We previously observed that the miR-106b-25 cluster inhibits anticancer drug-induced cell death in LNCaP cells. We think that miR-106b is the mediator of the anti-apoptotic effects of the miR-106b-25 cluster and designed experiments to identify the apoptosis genes that are inhibited by miR-106b. A recent publication (Proc Natl Acad Sci USA 105:10513-18, 2008) found that circulating microRNAs are stable blood-based markers that may prove useful for cancer detection. We have begun evaluating the technology for analyzing microRNA abundance in plasma samples from cases and controls to examine whether the microRNA expression profile of blood samples is predictive for cancer diagnosis and prognosis. In a pilot study, we examined the relative abundance of several candidate microRNAs in plasma samples from cancer patients and population-based controls. These microRNAs were selected from the tumor microRNA expression profile that we identified (mir-32, -106b, -141, -182, -196a, -199a, -200a, -200c). Preliminary findings indicate that most microRNAs are at the detection limit using our current protocol of total RNA isolation and pre-amplification for the qRT-PCR assay. Future work is aimed to improve our protocol, e.g., removing heparin from our samples, and adding EGTA. Discussions with colleagues revealed that heparin, Ca, and Mg may inhibit the microRNA assays.
