Upregulation of miR-3195, miR-3687 and miR-4417 is associated with castration-resistant prostate cancer.

PURPOSE: Prostate cancer (PCa) is a leading cause of cancer-related death. Upon androgen-deprivation therapy, the disease may progress further to castration-resistant PCa (CRPC) with a poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs, which play crucial roles in gene regulation. The aim of our study is to find CRPC-associated miRNAs and to evaluate their functional role. METHODS: In this study, 23 benign prostatic hyperplasia (BPH), 76 primary PCa, and 35 CRPC specimens were included. Total RNA extracted from tissue sections was used for miRNA profiling on the Affymetrix GSC 3000 platform. Subsequently, stem-loop RT-qPCR analysis was performed to validate the expression levels of selected miRNAs. PCa cell lines were transfected with miRNA mimics or inhibitors to evaluate the effects on cell proliferation, cell migration and cell invasion. RESULTS: In our profiling study, several miRNAs were found to be deregulated in CRPC compared to primary PCa tissue, of which miR-205 (- 4.5-fold; p = 0.0009), miR-92b (- 3.1 fold; p < 0.0001) were downregulated and miR-3195 (5.6-fold; p < 0.0001), miR-3687 (8.7-fold; p = 0.0006) and miR-4417 (5.0-fold; p = 0.0005) were most upregulated. While KLK3, miR-21 and miR-141 expression levels in androgen-treated VCaP and LNCaP cells were increased, the expression levels of miR-3687 and miR-4417 were reduced. None of the miRNAs were androgen-regulated in the AR-negative PC3 cell line. Overexpression of miR-3687 reduced cell migration and cell invasion, whilst miR-3195 enhanced cell migration. CONCLUSION: We have identified several novel deregulated miRNAs in CRPC tissue, including two microRNAs that are potentially involved in tumor invasion. Our data support the hypothesized involvement of miRNAs in PCa tumorigenesis and progression to CRPC. The applicability of these miRNAs as novel biomarkers for CRPC remains to be further investigated.

DD3: a new prostate-specific gene, highly overexpressed in prostate cancer.

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in the Western male population. Despite the tremendous efforts that have been made to improve the early detection of this disease and to design new treatment modalities, there is still an urgent need for new markers and therapeutic targets for the management of prostate cancer patients. Using differential display analysis to compare the mRNA expression patterns of normal versus tumor tissue of the human prostate, we identified a cDNA, DD3, which is highly overexpressed in 53 of 56 prostatic tumors in comparison to nonneoplastic prostatic tissue of the same patients. Reverse transcription-PCR analysis using DD3-specific primers indicated that the expression of DD3 is very prostate specific because no product could be amplified in 18 different normal human tissues studied. Also, in a sampling of other tumor types and a large number of cell lines, no expression of DD3 could be detected. Molecular characterization of the DD3 transcription unit revealed that alternative splicing and alternative polyadenylation occur. The fact that no extensive open reading frame could be found suggests that DD3 may function as a noncoding RNA. The DD3 gene was mapped to chromosome 9q21-22, and no homology of DD3 to any gene present in the computer databases was found. Our data indicate that DD3 is one of the most prostate cancer-specific genes yet described, and this makes DD3 a promising marker for the early diagnosis of prostate cancer and provides a powerful tool for the development of new treatment strategies for prostate cancer patients.

The genes for the calcium-dependent cell adhesion molecules P- and E-cadherin are tandemly arranged in the human genome.

Cadherins constitute a gene family of Ca(2+)-dependent cell-cell adhesion molecules involved in the morphogenesis and maintenance of tissue integrity. E- and P-cadherin are members of the cadherin family that are both expressed in epithelial tissues. Here we present the localization of the human P-cadherin gene at 32 kb upstream of the human E-cadherin gene, mapping it to chromosome 16q22.1. Tandem arrangement of two cell-cell adhesion molecules from the cadherin family has also been reported in chicken. This is the first evidence for the direct linkage of two cadherins in mammals. The evolutionary conservation of the tandem arrangement of two genes encoding cell adhesion molecules suggests that the close proximity of the genes may be important for the regulation of the genes.

A system to determine basepair substitutions at the molecular level, based on restriction enzyme analysis; influence of the muc genes of pKM101 on the specificity of mutation induction in E. coli.

A system has been developed for the analysis of basepair substitutions that are involved in the reversion of a specific missense mutation. The method is based on the ability of restriction enzymes to recognize and cut specific DNA sequences. Wild-type revertants arising from AT----GC transitions, pseudo wild-type revertants arising from AT-transversions and second site revertants can be distinguished. 4 mutagenic agents have been used, 2,6-diaminopurine, MMS, EMS and ENU, which differ in the types of damage they cause in DNA and in the susceptibility of the damage to repair. All 4 mutagens effectively enhanced the reversion of the mutation studied, trpA223, particularly by increasing the fraction of AT----GC transitions. In this system the influence of the muc genes of plasmid pKM101 was investigated. The presence of these genes reduced the fraction of AT----GC transitions and enhanced the fraction of AT-transversions as well as the fraction of second-site mutations. This change in mutation specificity is found irrespective whether mutation induction occurs mainly via SOS repair (MMS, ENU) or via mainly misreplication (2,6-diAP, EMS). These data suggest that the muc genes are involved in the induction of mutations not only during SOS repair, but also during misreplication. The change in mutation specificity may be caused by a change in the selection and insertion of nucleotides by the DNA-polymerising complex, or by interference with the repair of mismatched bases.