Site directed mutagenesis of miRNA binding site on the 3′-UTR of choline kinase alpha gene
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 (chk...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://eprints.usm.my/58370/1/Bnisallama-24%20pages.pdf |
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| Summary: | 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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