Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase which
activates, upon EGFR binding, a number of signaling pathways including the mitogenic protein
kinase pathway (MAPK) and phosphatidylinositol 3-kinase cascade (PI3K). Over expression of
EGFR is a common feature in variety of human cancers including lung, colorectal, breast,
pancreatic and oesophageal cancers and results in autonomous cell growth, enhanced metastatic
potential, tissue invasion and increased resistance to current cancer therapeutics. Thus EGFR has
been identified as a potential target in cancer therapeutics. Using the RNA interference (RNAi)
pathway, the aim was to specifically knockdown expression levels of endogeneous EGFR in
human oesophageal squamous carcinoma cell (HOSCC) lines. The RNAi pathway was initiated
through the transfection of three specifically designed short hairpin RNAs (shRNAs) against
human EGFR. The shRNAs were specifically designed using bioinformatics tools and their
individual knockdown efficacy determined through the introduction of an exogeneous based
target reporter systems, psiCHECK and pcieGFP. Expression levels of EGFR were determined
using Western blot analysis followed by densitometry. Knockdown of EGFR was achieved by all
three EGFR shRNAs in the three HOSCC cell lines (WHCO1, WHCO5 and WHCO6) despite
low transfection levels of ~10%. Greastest knockdown of EGFR (85%) was achieved by EGFR
sh2 in WHCO5. EGFR sh2 and sh1 resulted in average knockdown of EGFR of ~ 65% in
WHCO1 and WHCO5 respectively. Weakest knockdown of EGFR (~ 20%) was obtained by all
three EGFR shRNAs following transfection of WHCO6. RNAi-based approaches therefore show
substantial potential for the specific and efficient targeting of EGFR in human cancer cells.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/4896 |
| Date | 27 May 2008 |
| Creators | Killick, Mark Andrew |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 2694674 bytes, 36632 bytes, 69756 bytes, 10956 bytes, application/pdf, application/pdf, application/pdf, application/pdf, application/pdf, application/pdf, application/pdf, application/pdf |
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