ESR Project 13: Regulating TRIB1 and TRIB3 Activity by miRNAs in macrophages
TRIB1 and TRIB3 have been recognized to play a fundamental role in regulating the phenotype and the function of immune cells, affecting monocytes and macrophages proliferation, differentiation and apoptosis.Also, the mRNAs of both genes have extended 3’untranslated regions and recent data suggested that miRNAs are important post-transcriptional regulators of TRIB genes.
The aim of this project is to discover and characterize miRNAs that regulate the fates of TRIB1 and TRIB3 mRNAs and proteins in monocytes and macrophages by using a combination of computational and experimental techniques. We will investigate tribbles target genes, including transcription factors and characterize the impact of immune-metabolic stimuli on TRIB action. The main objectives and the project workflow are outlined in Figure 1.
The first step of this study is the application of bioinformatics to analyse the publicly available monocyte/macrophage gene expression datasets, including RNA-seq, miRNA-seq and RNA microarrays data. We will also interrogate online miRNAs databases to identify both predicted and validated TRIB1-3/miRNAs interactions. A computational chemogenomics approach will also be applied to screen for small-molecules that may control TRIB expression, including a potential mechanism via miRNAs. In addition, Single Nucleotide Polymorphisms in the 3’ UTR regions will help us to identify the presence of isoform-specific binding activities.
Next, miRNA effects on TRIB1 and TRIB3 will be tested experimentally using Dual Luciferase Reporter Assay, in which the transcriptional activity of Firefly and Renilla luciferase reporters are sequentially measured from a single sample. We have created tribbles 3’UTR Reporter Vectors to evaluate the impact of tribbles 3’untranslated regions on mRNA stability and determine whether candidate miRNAs alter this. We will assess the impact of miRNAs on both tribbles RNA and protein expression by RT-qPCR and Western Blotting in primary human macrophages and monocyte/macrophage cell lines, by transfecting cells with mimics and inhibitors of candidate miRNAs. In this context, we will evaluate the impact of immune-metabolic stimuli on macrophage activity (e.g. polarization, cytokine expression profile, phagocytosis).
Finally, we will characterize the impact of tribbles regulator miRNAs using RNA/miRNA Specific Blockers. As miRNAs have multiple RNA targets, by using miRNA mimics or inhibitors we may interfere with the activity of multiple genes. Therefore, the use of RNA/miRNA Specific Blockers will provide us with a more specific experimental tool to characterize miRNAs that directly regulate TRIB expression.