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Characterisation of miR-494 in coagulation

Lui, Wing (2016) Characterisation of miR-494 in coagulation. Honours thesis, Murdoch University.

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Micro-ribonucleic acids (miRNAs) are non-coding RNAs that, in the majority of cases, regulate gene expression by binding to the 3’ untranslated region (UTR) sequence of target mRNAs to cause decreased mRNA levels. Recently, several studies showed that selected miRNAs also target important genes in coagulation and haemostasis. The miRNA, miR-494, downregulates Protein S (PS) mRNA levels by binding to the 3’UTR sequence of PROS1 mRNA transcript. Further study demonstrated that some coagulation factors without predicted miR-494 binding sites in their 3’UTR sequences showed significant changes in their mRNA levels, including plasminogen (PLG), tissue factor (F3) and C4BPα (C4BPA). It is possible that miR-494 directly targets transcriptional activators or repressors to indirectly regulate the expression of those coagulation factors lacking predicted miR-494 binding sites. This current study hypothesised that miR-494 has an important role in regulating coagulation pathways and haemostasis by targeting multiple coagulation factor genes via direct and indirect mechanisms. HuH-7 cells were transfected with miR-494 for 48h and 72h followed by mRNA and protein analysis. Direct interaction between miR-494 and transcription factor 3’UTRs (JUN, SP1 and STAT5B 3’UTRs) was determined using dual-luciferase reporter assays. The mRNA and protein levels of PS and PLG was significantly downregulated, and the C4BPA mRNA and protein levels were upregulated with the presence of miR-494. Moreover, the protein level of tissue factor (TF) was decreased at 72h post-transfection but no changes were found in its mRNA levels. Computational analyses showed that predicted miR-494 binding sites were found in the JUN, SP1 and STAT5B 3’UTRs. Dual luciferase reporter assay confirmed the presence of functional miR-494 binding sites in the 3’UTR sequence of SP1 and STAT5B. The SP1 and STAT5B mRNA levels were significantly downregulated with the presence of miR-494 but no change was observed for the JUN mRNA levels. Computational analysis showed that a predicted Sp1 binding site was found in the promoter region of human PLG gene. A report by Gutierrez-Fernandez et. al. (2007) suggested that Sp1 acts as a transcriptional activator in the murine PLG promoter. These suggested that miR-494 may indirectly downregulate PLG expression by targeting Sp1. Taken together, miR-494 directly downregulates PS expression, and indirectly downregulates PLG expression through Sp1 repression and upregulates C4BPA expression. These results suggested that miR-494 has a prothrombotic effect.

Publication Type: Thesis (Honours)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Tay, Jasmine and Mead, Robert
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