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Title: Discovery and characterisation of small RNAs in bone cancer
Author: Green, Darrell
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2016
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Regulation of gene expression is of fundamental importance to maintain organism function and integrity and requires a multifaceted and highly ordered sequence of events. Disruption of these events can result in significant costs such as genome instability, accelerated ageing and disease. Small RNAs are non-coding RNA molecules that are key regulators of gene expression at both the transcriptional and post-transcriptional level. Next generation sequencing of small RNA libraries is widely used for studying small RNAs in various biological systems. Small RNA libraries are biased for sequences that are more likely to anneal to adapters than other small RNA sequences. This bias is due to the complementarity or annealing efficiency between sequences and adapters. Our laboratory previously developed High Definition (HD) adapters which contain degenerate nucleotides at the ligating ends of the adapters. HD adapters reduce the bias by recovering sequences for analysis that would have been missed with standard kit adapters. Current commercial kits for small RNA library construction produce a large amount of 5’ adapter – 3’ adapter ligation products without the cDNA insert when HD adapters are used to replace standard kit adapters. The work here reports an improved method to generate small RNA libraries using HD adapters with significantly reduced adapter-adapter products. By using HD adapters in next generation sequencing and applying to a clinical association study, tRNA fragment (tRF) expression is modulated throughout chondrosarcoma progression. An additional characteristic of high grade tumours is the reactivation of a microRNA-mediated embryonic bone development pathway. Through loss-of-function and gain-of-function studies the work proposes tRF-Gly-TCC is a potent tumour suppressor in which treated tumour organoids display a unique cell morphology and changes in gene expression. This work also finds miR-16 is upregulated in Paget’s associated osteosarcoma. MiR-16 could be used as a biomarker for early diagnosis of the disease. MiR-16 as a marker of Paget’s associated osteosarcoma was confirmed in a study on an ancient sample of this cancer that we acquired from a collection of medieval skeletons at Norton Priory. The improved small RNA library method was further used in a benign bone tumour which showed the characteristics of a malignant bone tumour except for the lack of a high expression of miR-21. MiR-21 is the most upregulated microRNA in human cancer.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available