title: miR-155 targets Caspase-3 mRNA in activated macrophages.
abstract: 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. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan(TM) we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation 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. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, 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. Strikingly, 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, we report 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 inflammation.
authors.affiliation: a Department of Intensive Care and Intermediate Care , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
authors.forename: Rebecca
authors.initials: R
authors.lastname: De Santis
authors.affiliation: a Department of Intensive Care and Intermediate Care , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.|b Department of Internal Medicine III , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Liepelt
authors.affiliation: a Department of Intensive Care and Intermediate Care , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
authors.forename: Jana C
authors.initials: JC
authors.lastname: Mossanen
authors.affiliation: c Biochemistry Center Regensburg (BZR) , Laboratory for RNA Biology, University of Regensburg , Universitätsstrasse 31, 93053 , Regensburg , Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Dueck
authors.affiliation: a Department of Intensive Care and Intermediate Care , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Simons
authors.affiliation: b Department of Internal Medicine III , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Mohs
authors.affiliation: b Department of Internal Medicine III , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Trautwein
authors.affiliation: c Biochemistry Center Regensburg (BZR) , Laboratory for RNA Biology, University of Regensburg , Universitätsstrasse 31, 93053 , Regensburg , Germany.
authors.forename: Gunter
authors.initials: G
authors.lastname: Meister
authors.affiliation: a Department of Intensive Care and Intermediate Care , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
authors.forename: Gernot
authors.initials: G
authors.lastname: Marx
authors.affiliation: a Department of Intensive Care and Intermediate Care , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Ostareck-Lederer
authors.affiliation: a Department of Intensive Care and Intermediate Care , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Ostareck
mesh_terms: D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000235:genetics*; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D015723:Gene Library; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D000067996:RAW 264.7 Cells; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D051197:Toll-Like Receptor 4 / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D035683:MicroRNAs; C532623:Mirn155 microRNA, mouse; D012333:RNA, Messenger; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C505409:Casp3 protein, mouse; D053148:Caspase 3
keywords: Caspase-3 mRNA; LPS; TLR4; macrophage activation; miR-155; post-transcriptional regulation