Investigating positionally conserved long non-coding RNA functions during zebrafish development and human melanoma.

Abstract

LncRNAs (lncRNAs) are a subset of non-coding genes that do not code for proteins and comprise a large proportion of the human genome. The assessment of evolutionarily conserved functions lncRNAs is challenging due to the lack of sequence conservation between vertebrates. Despite this, lncRNAs are increasingly being recognised as crucial gene regulators in embryogenesis and cancer tumorigenesis. Melanoma is an aggressive skin cancer which derives from the malignant transformation of melanocytes and is associated with a poor prognosis once metastasised. Despite the promising potential of melanoma associated lncRNAs to generate new treatments, our understanding of their mechanisms which govern melanoma biology remains to be elucidated. The MITF and SOX10 transcription factors are essential for melanocyte development and melanoma biology regulating cancer associated processes such as proliferation and invasion. We identified human-zebrafish positionally equivalent melanoma and melanocyte expressed lncRNAs whose human orthologues are predicted to be regulated by MITF or MITF-SOX10. These lncRNAs hold promise for the development of new melanoma treatments, but first we must understand their functions in development and disease.
We identified 506 and 56 human melanoma and melanocyte expressed lncRNAs whose loci are bound by either MITF or MITF-SOX10, respectively, and that have a positionally equivalent zebrafish lncRNA transcript. We prioritised Differentiation Antagonizing Non-Protein Coding RNA (DANCR) and LINC00327 as examples of MITF and MITF-SOX10 regulated lncRNAs respectively. In this thesis a focus was placed on DANCR which is transcribed from syntenic regions in human and zebrafish genomes. We have shown that melanoma patients with high DANCR expression display decreased survival and that DANCR expression is regulated by oncogenes MITF and c-MYC. Consistently, depletion of the DANCR transcript reduces cell proliferation and migration of human melanoma cells. DANCR is a cytoplasmically-enriched regulator of genes involved in cell proliferation, migration, cell cycle and stress response. Zebrafish dancr is also cytoplasmically-enriched and is widely expressed in early development. CRISPR-mediated deletion of the zebrafish dancr promoter leads to a dorsalisation-like phenotype where tail structures are lost. This phenotype is partially rescued by co-injection of dancr RNA demonstrating that the dancr transcript is functional. We also show a potential developmental role for snora27, a snoRNA embedded within the intronic regions of dancr during zebrafish development. Human-zebrafish Dancr was also found to regulate expression of a key cell cycle regulator, Cdkn1a, suggesting a conserved function for Dancr. Together our work indicates that Dancr is a conserved lncRNA oncogene that is crucial during early vertebrate development.

Date of Award	7 May 2025
Original language	English
Awarding Institution	University of Bath
Supervisor	Keith Vance (Supervisor) & Robert Kelsh (Supervisor)