Bnisallama, Faisal Fayez Saffah
(2022)
Site directed mutagenesis of miRNA binding site
on the 3′-UTR of choline kinase alpha gene.
Masters thesis, Universiti Sains Malaysia.
Abstract
MicroRNA largely controls gene expression by attaching to messenger RNA (mRNA) in
the cell cytoplasm. Instead of being promptly translated into a protein, the targeted mRNA
will either be destroyed and its components recycled, or it will be retained and translated
later. Choline kinase alpha (chka) overexpression is a clinical sign of diseased tissues and
malignant cells. MicroRNAs (miRNAs) are effective posttranscriptional regulators of
gene. Studies by our team showed that these three miRNAs (miR-876-5p, miR-367-3p
and miR-32-5p) downregulated the expression of chka gene. However, the binding sites
of these miRNAs on the 3'-UTR of the chka gene have not been verified. This study aimed
to mutate the binding sites of these miRNAs for subsequent verification by a luciferase
assay. In this study, we performed PCR site-directed mutagenesis on the miR-367-3p
(GAAGCAGAAAT ATAGTGCAATA) from nucleotides (nt) 1817-1825 binding sites in
chka and miR-876-5p (GAG TGTAGCTGTG AAATCCA) binding site from nucleotides
(nt) 2573-2581 binding sites and verified the mutation by DNA sequencing. In vitro work,
after mutagenesis step, the PCR products were sent for sequencing, however the results
were not satisfactory.
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