Role of microRNAs in regulating endothelial cell functions

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The central hypothesis of this research program is that microRNAs (miRNAs) by regulating the expression of endothelial adhesion receptors such as E-selectin are major regulators of the adhesion of circulating cells to ECs. In particular, we hypothesize that miRNAs act in a feedback loop to repress the expression of E-selectin. We will pursue three specific objectives to test our hypothesis. 1) To ascertain the role of miRNAs in regulating cell adhesion and transendothelial cell migration. ECs will be transfected with mimics or inhibitors of miRNAs before being treated with inflammatory cytokines to induce the expression of E-selectin. Then, the adhesion of cells will be evaluated in static and dynamic conditions. Thereafter, the expression of miRNAs will be evaluated as well as their capacity to bind to the 3'UTR region of E-selectin mRNA. 2) To identify the signaling pathways triggering the expression of miRNAs. The involvement of the pathways known to regulate the expression of E-selectin in response to inflammatory cytokines will be evaluated. The approaches will rely on the use of chemical inhibitors, dominant negatives and siRNA-mediated knockdown of proteins of these pathways. 3) To investigate the mechanisms regulating the expression of miRNAs. The transcription level of pri-miRNAs will be determined. The processing of the pre-miRNAs and the stability of miRNAs will also be evaluated. The composition of the RNA-induced silencing (RISC) complex will be determined by appropriate cutting edge approaches in use in the lab. The role of phosphorylation of the RISC components will also be ascertained. Overall, this series of experiments will allow us to establish for the first time how miRNAs and miRNA-regulating pathways modulate E-selectin expression to modulate the adhesion of circulating cells to ECs. In corollary, our innovative research program will open new avenues in understanding the role of miRNAs in cell-cell adhesion and cell migration. Ultimately, our results will help to better understand inflammation, immunity and cancer.