Pradeep, Raikundalia Sweta
(2021)
Microrna regulation of human choline kinase alpha gene expression and function in cancer cell lines.
PhD thesis, Universiti Sains Malaysia.
Abstract
Choline kinase alpha (CHKA), the first enzyme of Kennedy pathway, has
noncatalytic function in tumour onset and progression. Overexpression of chka is a
clinical feature in malignant cells and cancerous tissues. microRNAs (miRNAs) are
efficient posttranscriptional regulators of gene expression. To date, the regulation of
chka gene expression by miRNAs has never been reported. The objective of this work
was to exploit the gene regulatory capabilities of chka-targeting miRNAs to
downregulate chka expression in breast (MCF7), cervical (HeLa) and liver (HepG2)
cancer cells and study the effects of the downregulated CHKA protein levels on
cellular processes. miRNAs exhibiting increased potential of interacting with the
3’UTR of chka gene were predicted using online available bioinformatic tools,
shortlisted based on predefined selection criteria and the shortlisted miRNA candidates
were tested in-vitro to study the miRNA:mRNA interaction in cancer cells. miR-32-
5p, miR-367-3p and miR-876-5p each exhibited strong in silico interaction with chka
mRNA, releasing free energy (MFE) lower than -1.00 kcal/mol and fulfilling other
predefined criteria for determining strong miRNA:mRNA interactions. Binding of the
selected three miRNAs with respective seed sites on 3’UTR of chka mRNA was
experimentally validated in MCF7, HeLa and HepG2 cells by luciferase assay. Effects
of miRNA:mRNA interaction on chka expression at mRNA and protein levels were
examined by qRTPCR and western blot. Cellular functions impacted by miRNA
mediated differential chka expression were studied by cell apoptosis assay, cell cycle
assay and wound healing assay. Compared to negative control, chka expression levels,
in HeLa, HepG2 and MCF7, as a result of transfection with miR-32-5p, miR-367-3p and miR-876-5p were 0.47-fold, 0.62-fold, and 0.76-fold, respectively in MCF7; 0.32-
fold, 0.38-fold, and 0.63-fold, respectively in HeLa; 0.44-fold, 0.49-fold and 0.51-fold,
respectively in HepG2 indicating maximum reduction was inflicted by miR-32-5p.
Similar effect was observed when miR-32-5p was combined with miR-876-5p, which
resulted in 0.33-fold chka downregulation in MCF7 cells. Correspondingly,
transfection of miR-32-5p also produced the strongest suppression of CHKA protein
levels in MCF7 (0.20-fold) and HeLa (0.46-fold) compared to other miRNAs tested.
The specificity of miRNA mimics towards chka was verified by the firefly luciferase
reporter assay and co-treatment with miRNA specific inhibitors, which led to reversal
of the decreased chka levels produced by the miRNAs. The three miRNAss and the
pair of miR-32-5p+miR-367-3p each initiated ~50% cells to undergo apoptosis in
HeLa and MCF7 cells. Maximum (77% of cell population) cell cycle arrest at the G0/G1
phase was prompted by miR-367-3p in MCF7. Wound repair function plummeted with
<50% wound area healed even after 96 hrs in MCF7, HeLa and HepG2 treated with
miR-32-5p alone and in combination with miR-367-3p. The data collected in this study
showed an inverse relationship between the three miRNA candidates and chka
expression level, thus, proving the potential of miR-32-5p, miR-367-3p and miR-876-
5p as biomarkers for early cancer prognosis and anti-cancer remedies in cancer types
with over expressed chka as a clinical observation.
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