In vitro and in vivo elucidation of the role of miRNAS in the apoptotic properties of BCL-XLsilenced human lung adenocarcinoma cells / Norahayu Othman
Anti-apoptotic BCL-XL is frequently overexpressed in non-small cell lung cancer, leading to inhibition of apoptosis and poor prognosis. MicroRNAs play a role in regulating apoptosis and cell survival during tumourigenesis, with cancer cells showing perturbed expression of miRNAs. The aim of this...
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| Format: | Thesis |
| Published: |
2017
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| Online Access: | http://studentsrepo.um.edu.my/7935/1/All.pdf http://studentsrepo.um.edu.my/7935/9/norahayu.pdf http://studentsrepo.um.edu.my/7935/ |
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| Summary: | Anti-apoptotic BCL-XL is frequently overexpressed in non-small cell lung cancer,
leading to inhibition of apoptosis and poor prognosis. MicroRNAs play a role in
regulating apoptosis and cell survival during tumourigenesis, with cancer cells showing
perturbed expression of miRNAs. The aim of this study was to determine the biological
effects of miRNA dysregulation on non-small cell lung cancer, and the molecular
mechanisms by which apoptosis is regulated. Overexpression and knockdown studies
were performed via transfection of miRNA mimics and inhibitors and cell death was
detected using the annexin V-FITC detection kit and caspase 3/7 activity assay. Cell
cycle analysis was also performed to determine the role candidate miRNAs play in cell
growth. Results indicated that overexpression of miR-608 and down-regulation of miR-
361-5p induced cell death in A549 and SK-LU-1 cells. Gene target prediction analysis
implicated various signaling pathways as targets of BCL-XL induced miRNA
alterations. Luciferase reporter assay identified AKT2 and SMAD2 as direct targets of
miR-608 and miR-361-5p, respectively, and suppression of its protein levels were
validated using Western blot. To elucidate the role and importance of these miRNAs in
vivo, labeled tumour cells were injected into the yolk sac of zebrafish embryos and
immunostained using monoclonal antibodies to detect the cleaved, active form of
caspase 3. In conclusion, BCL-XL silencing in A549 and SK-LU-1 cells leads to the
occurrence of apoptosis through the dysregulation of miR-608 and miR-361-5p, thus
providing a platform for anti-sense gene therapy whereby miRNA expression can be
exploited to increase the apoptotic properties in lung adenocarcinoma cells. |
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