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Title: The regulation of p53-dependent microRNA expression in response to genotoxic stress
Author: Purmessur, Nadia Sheree
ISNI:       0000 0004 5347 9967
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2014
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Introduction: miR-16 and miR-26a have been identified as key effectors of the p53 pathway in response to genotoxic stress. This work is focused on preliminary elucidation of regulatory mechanisms by which p53 controls expression of miR-16 and miR-26a and characterisation of their gene targets involved in the p53 network. Methods: Microarray expression analysis of miR-16 and miR-26a was followed by Q-PCR to confirm these miRNAs dependence on p53. We analysed the transcriptional regulation of these miRNAs by p53 via luciferase assay and ChIP assay. We investigated these miRNAs contribution to p53-dependent response to genotoxic stress. To validate miR-16 and miR-26a targets (Cyclin E, CHK1, and WEE1) we employed Q-PCR, western blotting and luciferase assay. We also analysed the transcriptional regulation of Cyclin E by SET9 via luciferase assay. Results: High miR-16 and miR-26a expression are associated with increased cancer survival. p53-dependent and -independent regulatory mechanisms exist for miR-16 and miR-26a, and p53 controls expression of miRNAs on several levels. p53 recruits Drosha complex to miR-16 and miR-26a to facilitate the processing of these miRNAs. miR-26a cooperates with p53 to induce apoptosis and miR-16 enhances p53-mediated cell cycle arrest. miR-16 and miR-26a regulates CHK1 and WEE1, in the presence or absence of p53. miR-16 also reduces Cyclin E levels, in the presence and absence of p53. SET9 controls expression of Cyclin E on a transcriptional level. Conclusions: Our results showed that in response to DNA damage, miR-16 and miR-26a expression levels are controlled by p53-dependent and p53-independent mechanisms, potentially involving other stress-response transcription factors such as E2F1. Our data also confirms that miR-16 and miR-26a directly target Cyclin E, CHK1, and WEE1 for down-regulation. Additonally, SET9 directly controls Cyclin E expression. Reduced CHK1 and WEE1 levels leads to decreased G2/M arrest, and reduced Cyclin E levels results in increased G1/S arrest. As a consequence, apoptosis occurs.
Supervisor: Barlev, Nickolai Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available