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Title: The role of the 3' untranslated region of tissue factor mRNA in its post-transcriptional regulation
Author: Cao, Jun
ISNI:       0000 0004 7657 0093
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Tissue factor (TF), an initiator of blood coagulation cascade, is not expressed in resting monocytes/macrophages but can be induced by LPS, providing a link between sepsis and thrombosis. In contrast, TF is highly expressed constitutively by MDA-MB-231 breast cancer cells. Our group has previously demonstrated that the stability of LPS-induced TF mRNA in macrophages is regulated by a complex involving the RNA binding proteins tristetraprolin (TTP) and poly (ADP-ribose)-polymerase-14 (PARP-14) and an AU-rich element (ARE) at the distal end of TF mRNA 3'UTR mRNA. In the present work we sought to continue this work by determining post-transcriptional regulatory mechanisms acting on constitutive TF expression in MDA-MB-231 cells. We confirmed high steady state levels of TF mRNA. Using a luciferase-TF 3'UTR reporter assay, we found that mutation of the distal ARE implicated in TTP/PARP-14 binding led to a significant increase in reporter activity. As TTP was not detectable in MDA-MB-231 cells, we sought other proteins that regulate TF by interacting with the ARE, and identified human antigen R (HuR). First, siRNA knock-down of HuR led to significant increase in luciferase-TF 3'UTR activity; secondly, we were able to western blot HuR in material pulled down with in vitro-transcribed TF 3'UTR, and this was significantly reduced using a construct with a mutation in the distal ARE. As HuR siRNA knock-down increased TF protein expression without detectably affecting TF steady state mRNA, we conclude that HuR probably plays a role in TF translational repression. Consistent with this, association of HuR with TF 3'UTR in the in vitro pull down assay fell after activation of macrophages with LPS, in parallel with the induced expression of TF and TTP.
Supervisor: Haskard, Dorian ; Johns, Michael Sponsor: Imperial College London ; Chinese Scholarship Council ; British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral