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Title: Using RNA aptamers to manipulate the functions of human papillomaviruses (HPV)
Author: Cesur, Ozlem
ISNI:       0000 0004 5363 6213
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2015
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HPV is the most common viral infection of reproductive tract, affecting both females and males. High-risk oncogenic types are the cause of cervical cancer by the expression of E6 and E7 viral oncoproteins. High-risk oncogenic types are also responsible for up to 90% of anal and 12 % of oropharyngeal cancers. The well-characterised interaction of HPV16 E7 is with the cell cycle control protein pRb, promoting pRb degradation and resulting in cell cycle misregulation. E7 can interact with many other cellular proteins. In this study, RNA aptamers identified against HPV16 E7 were used to study E7 interactions further. The aptamers were generated previously by an in vitro technique known as the systematic evolution of ligands by exponential enrichment (SELEX), in which molecules from a random pool are isolated for binding to the target with a high affinity and specificity. Aptamers are single-stranded oligonucleotides that can form complex structures and bind target molecules in a conformation-dependent manner. The E7 aptamers have been stabilised by the inclusion of modified pyrimidines. Aptamers have therapeutic potential; examples include the aptamer Macugen (also called Pegaptanib). Several HPV16 E7 aptamers were able to induce apoptosis in an HPV16-transformed cervical carcinoma cell line (SiHa) that actively express both E6 and E7. Of particular interest is the E7 aptamer A2. In order to ensure apoptosis is not due to the innate immune response, immune stimulatory effects of E7 aptamers were studied by quantitative PCR. Effects of aptamers on the steady state levels of E7 and cellular proteins were analysed by western blotting. Treatment of cells with A2 resulted in loss of the E7 oncoprotein and a rise in cellular pRb levels. We have evidence that some of the HPV16 E7 aptamers can also target HPV18 E7. Several inhibitor molecules of protein degradation were used to determine the pathway of E7 loss following aptamer transfection in HPV16-transformed cervical carcinoma cell line, CaSki. Results suggested that aptamer transfection did not appear to alter normal degradation pathway of E7, which was mainly 26S proteasome-mediated. Co-localisation of aptamers (model aptamers) with cellular markers was studied by immunofluorescence. Co-localisation of E7 with cellular markers in the presence or absence of HPV16 E7 aptamers suggested the accumulation of E7 in the endoplasmic reticulum (ER) in A2 transfected cells, suggesting an alternative pathway, leading to a model for E7 degradation. Aptamers can be advantageous in terms of developing novel potential therapeutics. In Chapter 5, model aptamers were shown to internalise into keratinocytes and cervical cancer cells without the need for lipofection. This novel ability of aptamers might enable topical use of aptamers. The mechanism of aptamer entry was also studied using endocytosis inhibitors for receptor-mediated uptake as well as macropinocytosis. The result suggested that reagent-free internalisation can be both energy-dependent (receptor-mediated) and passive (macropinocytosis) and that uptake can be sequence-dependent.
Supervisor: Stonehouse, Nicola J. ; Blair, G. Eric Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available