Aim: NOD1 and 2 are intracellular pattern recognition receptors mediating vascular inflammatory processes. Since endothelial cells are crucially involved in vascular inflammation, we here investigated the microRNA (miRNA) expression profile in primary murine endothelial cells upon NOD stimulation and their role in endothelial cytokine expression. 

Methods: Primary lung endothelial cells were isolated from C57BL/6 mice (CD31/CD144), expanded and subjected to purity control (flow cytometry). Cells were stimulated with Tri-DAP (NOD1) or MDP (NOD2) respectively for 6h followed by RNA isolation. After RNA quality control, miRNA expression profiling was assessed by TaqMan Low Density Array. Raw data were analyzed using R Package HTqPCR (Bioconductor). Significantly regulated microRNAs were validated by qPCR and in-silico target gene analysis was performed (DIANA/miRPath, TargetScan, miRDB, miRTarBase). Target genes were analyzed performing miRNA transfection experiments and luciferase assays.

Results: Array analysis revealed 10 NOD-dependently regulated miRNAs (P<0.05; FC >±1.5). Two candidates, miR-147-3p and miR-298-5p, were validated by qPCR (P<0.05) in wildtype and NOD1/2-deficient primary endothelial cells. MiR-298-5p was found to be down-regulated by NOD1 stimulation and miR-147-3p was found to be down-regulated after both NOD1 and NOD2 stimulation. In-silico analysis revealed target genes involved in inflammatory pathways as TNF-α and IL-6, which were successfully validated by transfection experiments for miR-298-5p and miR-147-3p. Luciferase assays revealed direct binding of miR-298-5p to IL-6 3´UTR. 

Conclusion: Here we firstly report NOD-dependently regulated miRNAs involved in vascular inflammation. Noteworthy, miR-147-3p and miR-298-5p target TNF-α and IL-6, thereby operating as NOD-driven amplifiers of cytokine induction.

03 - MICRORNAS MIR-147-3P AND MIR-298-5P ARE NOD-DRIVEN REGULATORS OF ENDOTHELIAL CYTOKINE EXPRESSION