Interrogation of structural variations of pro-oncogenic AGR2 protein in supporting esophageal cancer progression / Nurul Hanani Baderol Hisham
A non-hormone-dependent cancer, esophageal adenocarcinoma was classified as the sixth deadliest cancer with an elevated mortality rate resulting from failure detection at an onset of the disease hence causing the tumor to be at either an advanced or metastatic stage upon diagnosis. Such cancer pr...
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| Format: | Thesis |
| Published: |
2022
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| Online Access: | http://studentsrepo.um.edu.my/14726/2/Nurul_Hanani.pdf http://studentsrepo.um.edu.my/14726/1/Nurul_Hanani.pdf http://studentsrepo.um.edu.my/14726/ |
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| Summary: | A non-hormone-dependent cancer, esophageal adenocarcinoma was classified as the sixth
deadliest cancer with an elevated mortality rate resulting from failure detection at an onset
of the disease hence causing the tumor to be at either an advanced or metastatic stage
upon diagnosis. Such cancer progression initially reported to be due to many factors
including the emergence of pro-oncogenic protein such as the AGR2 protein, known to
play roles mainly in the folding of proteins as well as maintaining proteostasis in the ER.
The poor understanding on the structural functions of the AGR2 protein in supporting
esophageal cancer progression has led to the design of this research which involved
constructing four AGR2 protein variants comprised of the AGR2 – WT variant as to
compare the basal expression to the other mutated variants, the AGR – KDEL variant to
enhance localization of the protein in the ER, the mAGR2 – ΔKTEL variant to enhance
secretion of the protein into the extracellular environment and the ΔNterm AGR2 – KDEL
variant to enhance the dimerization of the protein. All constructs of AGR2 protein which
were AGR – WT, AGR2 – KDEL, mAGR2 – ΔKTEL and ΔNterm AGR2 – KDEL were
cloned into the pSF-CMV vector before basal expression of the AGR2 protein variants
were determined by transiently transfecting them into the FLO-1 esophageal
adenocarcinoma cell line and conduction of immunoblotting and immunofluorescence.
The intracellular expressions of the FLO-1 showed the highest expression observed by
the AGR2 – WT variant followed by AGR – KDEL. Such expressions were further
validated by immunofluorescence. Reduced extracellular expressions of the AGR2
variants in FLO-1 were observed only in two variants, the AGR2 – WT and the AGR –
KDEL, while no extracellular expression was observed for the mAGR2 – ΔKTEL variant. The expressions of the ΔNterm AGR2 – KDEL variant either intracellularly or
extracellularly could not be validated either by Western blot or immunofluorescence due
to the lack of transfection efficiency. The accession on the influence of the AGR2 protein
variants towards proliferation and migration of the FLO-1 observed from wound healing
assay revealed significant elevations for FLO-1 proliferation and migration in AGR2 –
WT, AGR2 – KDEL and mAGR2 – ΔKTEL (p<0.05). The viability of the FLO-1
transfected to AGR2 – WT, AGR2 – KDEL and mAGR2 – ΔKTEL through conduction
of MTT assay revealed a significant result (p<0.05) supported by an increase in the
absorbances of the cells with respect to time in contrary to Alamar Blue assay conducted
which revealed an insignificant result (p>0.05) suggesting that the transfected AGR2
protein variants were independent towards the viability of the FLO-1.
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