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

Androgen signalling through the androgen receptor (AR) is essential for prostate cancer initiation, progression and transformation to the lethal castration-resistant state. The aim of this study was to characterize the mechanisms by which miR-145 deregulation contribute to prostate cancer progression. The miR-145 levels, measured by quantitative reverse transcription-polymerase chain reaction, were found to inversely correlate with occurrence of metastases, survival and androgen deprivation therapy response in a well-characterized prostate cancer cohort. Introduction of ectopic miR-145 in prostate cancer cells generated an inhibitory effect on the AR at both transcript and protein levels as well as its activity and downstream targets prostate-specific antigen (PSA), kallikrein-related peptidase 2 and TMPRSS2. The regulation was shown to be mediated by direct binding using Ago2-specific immunoprecipitation, but there was also indication of synergetic AR activation. These findings were verified in clinical prostate specimens by demonstrating inverse correlations between miR-145 and AR expression as well as serum PSA levels. In addition, miR-145 was found to regulate androgen-dependent cell growth in vitro. Our findings put forward novel possibilities of therapeutic intervention, as miR-145 potentially could decrease both the stem cells and the AR expressing bulk of the tumour and hence reduce the transformation to the deadly castration-resistant form of prostate cancer

Rapidly evolving marmoset MSMB genes are differently expressed in the male genital tract

BACKGROUND: Beta-microseminoprotein, an abundant component in prostatic fluid, is encoded by the potential tumor suppressor gene MSMB. Some New World monkeys carry several copies of this gene, in contrast to most mammals, including humans, which have one only. Here we have investigated the background for the species difference by analyzing the chromosomal organization and expression of MSMB in the common marmoset (Callithrix jacchus). METHODS: Genes were identified in the Callithrix jacchus genome database using bioinformatics and transcripts were analyzed by RT-PCR and quantified by real time PCR in the presence of SYBR green. RESULTS: The common marmoset has five MSMB: one processed pseudogene and four functional genes. The latter encompass homologous genomic regions of 32-35 kb, containing the genes of 12-14 kb and conserved upstream and downstream regions of 14-19 kb and 3-4 kb. One gene, MSMB1, occupies the same position on the chromosome as the single human gene. On the same chromosome, but several Mb away, is another MSMB locus situated with MSMB2, MSMB3 and MSMB4 arranged in tandem. Measurements of transcripts demonstrated that all functional genes are expressed in the male genital tract, generating very high transcript levels in the prostate. The transcript levels in seminal vesicles and testis are two and four orders of magnitude lower. A single gene, MSMB3, accounts for more than 90% of MSMB transcripts in both the prostate and the seminal vesicles, whereas in the testis around half of the transcripts originate from MSMB2. These genes display rapid evolution with a skewed distribution of mutated nucleotides; in MSMB2 they affect nucleotides encoding the N-terminal Greek key domain, whereas in MSMB3 it is the C-terminal MSMB-unique domain that is affected. CONCLUSION: Callitrichide monkeys have four functional MSMB that are all expressed in the male genital tract, but the product from one gene, MSMB3, will predominate in seminal plasma. This gene and MSMB2, the predominating testicular gene, have accumulated mutations that affect different parts of the translation products, suggesting an ongoing molecular specialization that presumably yields functional differences in accessory sex glands and testis

Structure, evolution and expression of glandular kallikrein genes

Prostate-specific antigen (PSA) is a worldwide used tool for the diagnosis and monitoring of prostate cancer. It is a serine protease and belongs to the family of glandular kallikreins. Humans have three classical glandular kallikreins, but it has been reported that rodents have many more. Lately, the family has been expanded to contain 12 new genes, KLK4–KLK15, that are situated at the same locus on 19q13.3–13.4 in humans. In our phylogenetic analysis, we were able to demonstrate that the classical glandular kallikrein genes can be considered a subgroup of the expanded glandular kallikrein family. The tissue kallikrein gene has 14 functional paralogs in the mouse and 10 in the rat, but there is only a single copy in artiodactyls, dogs and primates. The arginine esterase in the canine prostate is the only identified functional ortholog of the progenitor to human PSA and hK2. In contrast, KLK4–KLK15 are conserved in the mouse and rat, suggesting that they can be used as model systems to elucidate the physiological importance of these genes. We found that AREIII in the distal promoter region of KLK2 related genes contained deletions in all pseudogenes, whereas it is intact in all functional genes investigated in the present study, which implies that such deletions may represent a step in the process leading to the silencing of KLK2 genes. Repair of the AREIII in the cotton-top tamarin KLK2H2 pseudogene, led to restoration of full transcriptional activity, confirming the importance of this element. Both PSA and hK2 were found to be expressed in many non-prostatic tissues, but at much lower levels than in the prostate. Furthermore, it was shown that PSA in ileum exhibits comparable proteolytic activity and potential to form complexes with protease inhibitors as does PSA in prostate

Non-coding RNAs in Prostate Cancer: From Discovery to Clinical Applications.

Prostate cancer is a heterogeneous disease for which the molecular mechanisms are still not fully elucidated. Prostate cancer research has traditionally focused on genomic and epigenetic alterations affecting the proteome, but over the last decade non-coding RNAs, especially microRNAs, have been recognized to play a key role in prostate cancer progression. A considerable number of individual microRNAs have been found to be deregulated in prostate cancer and their biological significance elucidated in functional studies. This review will delineate the current advances regarding the involvement of microRNAs and their targets in prostate cancer biology as well as their potential usage in the clinical management of the disease. The main focus will be on microRNAs contributing to initiation and progression of prostate cancer, including androgen signalling, cellular plasticity, stem cells biology and metastatic processes. To conclude, implications on potential future microRNA-based therapeutics based on the recent advances regarding the interplay between microRNAs and their targets are discussed

Two-Tailed RT-qPCR for the Quantification of A-to-I-Edited microRNA Isoforms

MicroRNAs are short non-coding RNAs with important functions in the regulation of gene expression in healthy and diseased tissues. To optimally utilize the biological and clinical information that is contained in microRNA expression levels, tools for their accurate and cost-effective quantification are needed. While the standard method, qPCR, allows for quick and cheap microRNA quantification, specificity is limited due to the short lengths of microRNAs and the high similarity between closely related microRNA family members. A-to-I editing can further diversify the microRNA pool by altering individual nucleotides. There is currently a lack of protocols for the accurate quantification of A-to-I-edited microRNA isoforms using qPCR. Here, we describe a protocol to quantify microRNA editing isoforms using two-tailed RT-qPCR, with either SYBR Green or hydrolysis probes. The user will perform reverse transcription of RNA samples, generate standard curves, and quantify the resulting cDNA in the following qPCR step. We also give guidelines for primer design and for the evaluation of assays using synthetic oligonucleotides. These tools are expected to be transferable to any A-to-I-edited microRNA and its isoforms

Semenogelin II gene is replaced by a truncated line 1 repeat in the cotton-top tamarin

The human seminal vesicles secrete two proteins, semenogelin I and semenogelin II, at very high concentrations. It has previously been shown that the cotton-top tamarin (Sanguinus oedipus), a New World monkey, is lacking the semenogelin II gene. We have now determined the nucleotide sequence of DNA located 5--13 kilobases (kb) downstream of the tamarin semenogelin I gene---a region that in man is occupied by the semenogelin II gene. Two regions with homology to the human semenogelin II gene were identified in the tamarin DNA. The first region, of 3.5 kb, is homologous to DNA upstream of the human gene, and the second region, of 0.6 kb, is mainly derived from the second intron. Between these regions, equivalent to 594 base pairs (bp) upstream of the transcription initiation site to 12 bp downstream of the stop codon in the human semenogelin II gene, the cotton-top tamarin DNA carries a truncated LINE1 repeat. In another set of experiments, the tamarin DNA hybridizing to the mouse semenoclotin gene was investigated. It was concluded that hybridization is with the second intron of the semenoclotin gene, but very likely, the material does not represent a cotton-top tamarin semenoclotin gene. Thus, a mammalian ancestor probably carried a single gene that in the rodent lineage developed into the semenoclotin gene and in the primate lineage into a progenitor of the semenogelin genes