Charakterisierung von miRNAs als Modulatoren des TLR4-abhängigen MAPK Signalwegs

To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. The expression of the kinases and their modulators involved in these pathways is regulated both at the transcriptional and post-transcriptional level. The aim of this work was to elucidate molecular mechanisms by which microRNAs (miRNAs) regulate target mRNA translation and stability in an inflammation-dependent manner.In order to identify differentially expressed miRNAs, cDNA libraries generated from untreated or LPS-activated murine macrophages (RAW 264.7) were analyzed by deep sequencing. This study focused on miRNA-155 (miR-155) that is highly induced by LPS. Northern blot and qPCR experiments confirmed the significant induction of miR-155 expression, which was detected in the cloning analysis. Employing TargetScanTM, CASPASE-3 (CASP-3) mRNA encoding a key player in apoptosis was identified as potential target of LPS-induced miR-155. LPS-dependent activation of primary murine macrophages revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. In order to prove a functional relation between miR-155 and CASP-3 mRNA Argonaute 2 (Ago2) immunoprecipitation was performed. Co-precipitation of miR-155 and CASP-3 mRNA demonstrated their accumulation in miRNA-induced silencing complexes (miRISC). Remarkably, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, this work revealed that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in the inflammatory response.IKKε represents an important protein of the TLR4 signaling pathway and the corresponding mRNA was published as a miR-155 target. However, ectopic miRNA-155 overexpression or inhibition did not clearly elucidate the mechanism underlying miR-155 mediated IKKε repression. This work showed that in contrast to CASP-3 mRNA, IKKε mRNA is rather translationally inhibited by LPS-induced miR-155 than destabilized