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Determination of the utility of oligonucleotide sequences specifically designed to disrupt the interaction of microRNA-494 with the PROS1 mRNA transcript

Rocchi, Alyssa (2017) Determination of the utility of oligonucleotide sequences specifically designed to disrupt the interaction of microRNA-494 with the PROS1 mRNA transcript. Honours thesis, Murdoch University.

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Embargoed until December 2018.

Abstract

Micro-ribonucleic acids (miRNA) are non-coding RNA molecules that function as negative regulators of cellular processes, for example gene expression, by binding to sites found within the 3’untranslated region (UTR) sequence of target messenger RNAs (mRNAs). A previous study identified miR-494 as a direct regulator of the PROS1 gene expression of Protein S (PS), with three functional binding sites being classified with the 3’UTR sequence. This regulation is a natural physiological process; however, it can become problematic for individuals with a hereditary PS deficiency. This current study hypothesised that the use of specifically targeted antisense oligonucleotide (ASO) sequences to the three functional binding sites found within PROS1 mRNA 3’UTR can disrupt, or block the binding of miR-494 resulting in a subsequent increased expression of PS. HuH-7 cells were cotransfected with miR-494 and ASOs and luciferase assay and mRNA analysis performed. Luciferase assay results showed a proof of principle when utilizing ASOs to block the interaction of miR-494 with the PROS1 3`UTR. The results were trending towards reversal for selected ASOs, however, not statistically significant. Subsequent mRNA analysis, did however demonstrate a clear significant reversal of the previously observed effect of miR-494 on the levels of PROS1 mRNA transcripts in the presence of the ASOs, when compared to miR-494 alone. Interestingly, further analysis suggested that key modifications designed to increase the stability and affinity of the ASOs was important for the potency of the blocking effects. Overall, this project provides preliminary evidence towards negating the regulation miR-494 has on the PROS1 3’UTR. These results are significant for individuals with a heterozygous mutation that is causative for a protein deficiency disease, as further testing can lead to a novel therapeutic agent that can bypass existing mutations, resulting in the increased expression of the remaining functional allele.

Publication Type: Thesis (Honours)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Hughes, Quintin and Adams, Murray
URI: http://researchrepository.murdoch.edu.au/id/eprint/39711
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