Please use this identifier to cite or link to this item: http://hdl.handle.net/1783.1/3917

Manipulation of EWS oncogene expression using RNAi

Authors Chan, Yuk Fai
Issue Date 2005
Summary The N-terminal of EWS Oncogene (EWS Activation Domain, EAD) is fused with different transcription factors by chromosomal translocation and formed a variety of EWS Fusion proteins (EFPs), which cause distinct humans sarcomas. EFPs can function as potent transcriptional activators, and de-regulation of cellular genes is thought to be a mechanism of tumorigenesis. The transcriptional properties of EWS/ATF1, one of the EFPs and specific in Clear Cell Sarcoma (CCS), has been extensively studied, but its biological role, like tumor maintenance, has not been studied seriously. The main reason is that a system to manipulate the expression of EWS/ATF1 has yet to be established. Therefore in this project, RNA interference (RNAi) was used to manipulate the expression of EWS/ATF1, and attempt to study its biological role in CCS. In this project, siRNA (IEA to IEE) against the EAD (target EWS/ATF1, as well as EWS) were used to manipulate the expression of EWS/ATF1 because it was suggested that EWS was a dispensable protein in sarcoma, so it was expected that knocking EWS would not affect the study of EWS/ATF1. However, after proving EAD siRNAs can knock down the expression of EWS/ATF1 and EWS, these siRNAs (IEA to IEC) could not be stably expressed in CCS cell (DTC1), as well as non-CCS cells (Jeg-3 and HeLa). This suggested that EAD siRNA is toxic to the cells, and EWS may be important for cell growth and maintenance. Other siRNAs had been tried to study EWS/ATF1, for example siRNA against the junction of EWS/ATF1 (siIEAj) and the C-terminal of ATF1 (siICA1 and siICA2), but none of them can carry out RNAi properly. Besides screening more siRNA against C-terminal of ATF1, EAD siRNAs could be still utilized to study EWS/ATF1. But the role of EWS in CCS should be clarified first. Therefore siRNA against C-terminal of EWS (siIRB) were tested, and can be used in the future in order to study the biological role of EWS in CCS. siIRB can distinguish the toxic effect of EAD siRNA in CCS, and it can show whether EAD siRNAs can be used to study EWS/ATF1 in CCS.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2005
Subjects
RNA
Language English
Format Thesis
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