Introduction Tribbles-1 (trib-1) pseudokinase is a regulatory protein that has been shown to be a protective gene in a number of cell types and processes, relevant to the development of atherosclerosis. These include inhibition of vascular smooth muscle cell proliferation, polarisation of macrophages towards an alternatively activated phenotype and lowering the production and release of LDL in hepatic tissues. Therefore, understanding the molecular mechanisms, which regulate trib-1 expression are of significant interest. Our group have identified miRNA202 as a controller of trib-1 levels.
Rationale Trib-1 mRNA is highly unstable with a half-life of less than 1 h; the 3’ UTR encodes for a number of putative binding sites for miRNAs, including miRNA202. This study aimed to experimentally validate the importance of miRNA202 in the control of trib-1 expression.
Methods 1. We have used a luciferase reporter to demonstrate the role of trib-1 3’UTR in mRNA stability and to characterise the impact of miR202 on trib-1 3’UTR in hepatic cells (HepG2). 2. qRT-PCR was used to quantify endogenous trib-1 and miRNA202 levels upon stimulation of IL-1 and in high glucose media. 3. Western blotting was used to elucidate the effect of miRNA202 on trib-1 protein levels.
Results 1. Overexpression of miR202 reduced luciferase – trib1-3’UTR reporter expression. 2. The endogenous trb-1 mRNA was also modulated by miR202. Furthermore, stimulation of IL-1 in HepG2 cells has shown trb-1 levels to reduce by >70% and miR202 levels to increase by 2 fold. 3. Trib-1 protein levels have shown a reduction upon overexpression of miRNA202. Conversely, miRNA202 inhibitor increased trib-1 protein levels.
Discussion miR202 is a novel regulator of trib-1 expression and may represent a target by which trib-1 levels could be raised in vivo, thereby providing a mechanism to augment the anti-atherosclerotic effects of this protein.