Objectives: My main objective in this study as a clinical master degree student was to reconstruct and expressed the wild and mutated-type (E545A) of p110α plasmids. Methodology: The wild-type of p110α plasmid was constructed by cloning the p110α gene (PIK3CA) from the normal cell genomic DNA by PCR.The clone p110α genes and the pcDNA3.1 plasmid were digested using the restriction enzymes Asc-1 and Sma-1.The digested fragments of p110α genes and pcDNA3.1 plasmid were then ligated using T4 DNA ligase.The recombinant DNA was then introduced into Escherichia Coli and finally transfected into HEK293A cells. The mutated-type (E545A) of p110α gene was induced using the QuickChange Lighting Site-Directed mutagenesis kit. Results: We successfully reconstruct and expressed the wild-type of p110α plasmid.The correct nucleotides sequence of the PIK3CA gene was confirmed by genesequencing.Overexpression of the p110α proteins in HEK293A were confirmed by western blot. The E545A mutation was successfully induced and confirmed by genesequencing.Downstream upregulation of the PI3K pathway in transfected HEK293 Aharbouring mutated p110α gene was confirmed by western blot showing overexpression of the AKT/PKB level. Discussion and Conclusion: The PIK3CA gene is mutated on average in 15％ of human cancers with mutations clustering predominantly within three hotspots: E542K,E545Kand H1047R. The E545A mutation although not considered as a hotspot mutation have been reported to contribute up to 88％ of the total of PIK3CA mutation identified in ovarian cancers and associated with various other types of cancers. We found in our department that 33％(36 cases out of 110) of patients with Gastrointestinal Stromal Tumour (GIST) also harbours this mutation.We strongly believed that tumour cells harbouring both KIT and E545A mutations are resistant to the receptor tyrosine kinase inhibitor-Imatinib.The reconstructed plasmids will be used in studying Imatinib resistance in GIST.
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