Genomics investigation into the regulatory network of TRIB1

Advances in TRIB1 protein research reveal its potential as a biomarker for various disease diagnoses, including cancer and atherosclerosis. TRIB1 is also known as highly unstable transcript with a half-life of less than one hour, making its functional regulatory network study difficult and therefore, remains unexplored. The first part of thesis focused on understanding the regulatory network of TRIB1. In 28 different cancer datasets coexpression analysis, revealed approximately 65% of genes coexpressed with TRIB1 belong to the immediate-early response (IER) gene family. EGR1 and FOS known IER genes were present in the coexpression module in 18/28 datasests. Furthermore in-vitro analysis suggested a relationship between EGR1, FOS and TRIB1. RNA-seq analysis from early response gene stimulated TRIB1 OE prostate cancer and control (DU145) at different time-points showed effects on many of the coexpressed IER genes, Differentially expressed genes were involved in cell signaling, cell proliferation and apoptosis. Further suggested, TRIB1 could be responsible for activating genes involved in several different cellular pathways, particularly the IER pathway andTRIB1 as a member of the early response gene family, Further study focused on understanding the post-transcriptional regulation of TRIB1 by identifying variants in the 3’ UTR and their effect on miRNA binding sites The results suggests that variants in 3’ UTR of TRIB1 are responsible in creating new miRNA binding sites, but they were not linked to the allele-specific expression, as would be expected if they were regulatory. The potential reasons being expression pattern of TRIB1: TRIB1 expression follows an IER pattern and degrades after one hour of cell-stress condition. In addition, novel binding sites for miRNAs generated in the TRIB1 UTR as variants are not mostly expressed in unstimulated and M1-like macrophages (LPS+ifg treated). We do however identify multiple other cases of gene variants in the 3’ UTR displaying allelic imbalance in their expression. Further study focused on understanding the role of miRNAs in macrophage polarization, nine hub miRNAs found to play an important role. For further validation, the expression of miR-125a-3p and miR-186-5p, with positive 13 control miR-155-5p were stimulated in unstimulated macrophages. RNA-seq analysis from miRNAs experiments reveals that target genes of miR-186-5p were more downregulated than the non-target ones unlike miR-125a-3p. The alternative polyadenylation analysis shows the unchanged length of 3’ UTR of target genes of miR-125a-3p between unstimulated and M1-like macrophages; stating no evidence of alternative polyadenylation phenomenon for miR-125a-3p target genes and miR-125a3p has less impact on degrading its target genes. Together these results shows that TRIB1 could a part of IER response gene network or TRIB1 is regulated by IER genes which in turn regulates the genes involved in cell-signaling, cell polarization pathways. Next, variants in 3’ UTR of TRIB1 are not linked to ASE, however, they are responsible in altering miRNA binding sites. That could be the reason of changes in expression level of TRIB1 in different cell-type. However, this needs to be investigated further. Furthermore, not all differentially expressed miRNAs between unstimulated and M1-like macrophages are important in macrophage polarization but the subset of those miRNAs i.e., nine (hub) miRNA plays an important role in macrophage polarization.