|dc.description.abstract||Cancer is a leading cause of mortality in developed countries. Due to the genetic and epigenetic heterogeneity of this disease, we still don’t have effective long-term therapies for many cancers. A characteristic of many cancer cells is an alteration in the structure of the nucleolus - the primary location of the ribosomal DNA (rDNA). The rDNA encodes ribosomal RNA, which is the major structural and catalytic component of ribosomes – the cellular machinery responsible for protein biosynthesis. Accordingly, the rDNA and its transcription is a key regulator of cell proliferation. Despite this critical role, the highly repetitive nature of the rDNA has made it difficult to study, thus it remains an attractive target for anti-cancer therapies. Indeed, the promising anti-cancer drug, CX-5461, developed by our collaborators, targets the rDNA through the inhibition of the rDNA dedicated RNA polymerase I (currently in clinical trials). In preliminary experimentation, there is a dramatic change in expression of non-coding RNAs (ncRNAs) from the rDNA during the transition to malignancy. Although the function of rDNA ncRNAs is almost entirely unknown, ncRNAs from other regions of the genome have a multitude of regulatory functions, including involvement in cancer. We hypothesise that these transcripts play a role in malignancy and CX-5461 sensitivity. Utilising a mouse B-lymphoma model (Eμ-myc), we first applied a high throughput hybridisation-based RNA-sequencing approach (capture-seq), to enrich for rDNA intergenic spacer (IGS) ncRNA transcripts within 11 cDNA sequencing libraries. Regions of transcription throughout the IGS were identified using several bioinformatic tools, and qPCR was performed to validate transcription status as well as assess for CX-5461-dependent transcriptional changes. We also utilised other bioinformatics tools, to predict small RNAs arising from the IGS and other regions of the Eμ-myc genome, and briefly assessed their response to CX-5461 treatment. miRNAs of interest were assessed for potential pathway targets using several bioinformatic targets. Lastly, we aimed to further characterise the Eμ-myc model. With this, we assessed efficacy of methods that could be used for downstream knockdown/over expression analysis.
Overall, using the capture-seq method we identified 8 major clusters of exons (known as exon cluster groups), that were consistently predicted between RNA library preparations. These were confirmed to be transcriptionally active by qPCR, with one of these clusters. Additionally, we identified several sites in the mouse rDNA IGS that may express small RNAs, with small RNA reads aligning to these sites with some consistency between library preparations. Some of these, due to presence and absence patterns in either CX-5461 treated or control libraries, may show some signs of treatment-dependent differential expression. We also identified miRNAs from other regions of the genome which show similar patterns. We assessed potential small RNAs for gene target enrichment. No pathways/cellular components appeared to be biologically significant. We assessed a method of viral-mediated gene knockdown in a number of cell lines, which did not show efficacy in the mouse lines we had available. In conclusion, if these exons produce ncRNAs that contribute to malignancy, the ncRNAs will form attractive new targets for therapy, independently or in combination with CX-5461, and could be used as diagnostic and prognostic markers of cancer. The future trajectories of this project include selecting promising IGS transcripts, particularly those differentially expressed, to confirm their size by northern blot. Then, to assess their role in malignant cells, to perform knockdown/overexpression assays and assess cellular response. Further, we would target the rDNA ncRNAs in several cancer and non-cancer cell lines, to broaden our understanding of anti-cancer application.||en_US