Identification of novel non-coding RNAs in prostate carcinoma cells

The human genome consists of approximately 3.3 billion nucleotides but only 1.5 % have protein coding features. Formerly, it was believed that the remaining nucleotides serve none of the known roles, they were considered as ""junk DNA"" and assumed to be useless. Nowadays it has been become obvious that there is an enormous part of this DNA being transcribed into RNA which do not code for proteins. In 2001 it was claimed that this applies to more then 97 % of the transcribed RNA [1]. These transcripts are so called non-coding RNAs (ncRNAs) and represent the main subject of this thesis. Recently, many studies show the abundance of several kinds of ncRNAs, for example long ncRNAs and micro RNAs. The main function of these transcripts is often unknown but their intracellular regulatory potential has been proven [2]. The importance of these transcripts becomes clear with the fact that it is probable that they are an essential part of eukaryotic physiological pathways and maintenance of cellular features. Many of them appear to contribute to the pathology of different types of cancer. However, their carcinogenic attributes and mechanisms remain in most cases unclear. In this project we aimed to identify and characterize novel ncRNAs in prostate cancer. This carcinoma can be distinguished into two different stages. The early stage of prostate cancer is an androgen dependent form and the second stage cancer cells are androgen independent. At the present day, there is one commonly used biomarker for this disease, the prostate specific antigen (PSA). Nevertheless, this marker is inaccurate and shows a high rate of false positive and a considerable number of false negative results [3]. Hence, there is a lack of accurate biomarkers to clearly distinguish between the two cancer stages and other prostate associated diseases. For the late stage of prostate carcinoma no curative therapies are available. Consequently, there is an urgent need for new more selective biomarkers and therapeutic targets. The association with cancer and often unknown physiological characteristics of ncRNAs lead to this new approach for the validation and characterization of suitable targets and markers. To find new potential ncRNAs, we compared the transcriptional activity of prostate tissue cell lines by the use of tiling array analysis. We restricted our view onto the chromosome 21 and 22 in three cell lines. RWPE-1 cells serve as a model for a benign prostate. The development from androgen-dependency to androgen-independency is an important characteristic during the development from early to late stage prostate cancer. In this project LNCaP cells represent an early stage prostate cancer model due to their androgen dependency. The PC3 cells function as a late stage cancer model because these cells are androgen independent. By means of statistical methods for the analysis of tiling array data in combination with available datasets 30 candidates for novel ncRNAs were retrieved. Ten candidates remained after further evaluations, using the integrated genome browser (IGB) from Affymetrix Inc. and considering their position in the genome. Five of the predicted candidates were tested. The experimental validation comprised reverse transcription PCR and quantitative expression measurement with SYBR-Green assays. Another cell line was included, DU-145 cells, as an additional model for the late stage cancer. From the 5 selected candidates 3 were validated. Two candidates on chromosome 21 are located in intronic regions the third candidate is localized in an intergenic region on chromosome 22. The expression patterns of the tested candidates represent the information obtained from tiling array experiments. All verified transcripts show the highest expression rate in LNCaP cells. Furthermore, experiments to determine the strand of origin were carried out, but did not lead to any determinations due to the occurrence of RNA priming. The expression levels and distribution of the candidate ncRNAs, between different conditions in different cell lines, as well as their possible physiological role need to be investigated further. However, this field is exceptionally promising in finding novel diagnostic markers and therapeutic targets, especially because their relevance and involvement in a rising number of diseases has been shown.