miR-155 targets Caspase-3 mRNA in activated macrophages

Rebecca De Santis; Anke Liepelt; Jana C Mossanen; Anne Dueck; Nadine Simons; Antje Mohs; Christian Trautwein; Gunter Meister; Gernot Marx; Antje Ostareck-Lederer; Dirk H Ostareck

doi:10.1080/15476286.2015.1109768

miR-155 targets Caspase-3 mRNA in activated macrophages

RNA Biol. 2016;13(1):43-58. doi: 10.1080/15476286.2015.1109768.

Authors

Affiliations

¹ a Department of Intensive Care and Intermediate Care , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
² b Department of Internal Medicine III , University Hospital, RWTH Aachen University , Pauwelsstrasse 30, 52074 , Aachen , Germany.
³ c Biochemistry Center Regensburg (BZR) , Laboratory for RNA Biology, University of Regensburg , Universitätsstrasse 31, 93053 , Regensburg , Germany.

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.

Keywords: Caspase-3 mRNA; LPS; TLR4; macrophage activation; miR-155; post-transcriptional regulation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Apoptosis
Caspase 3 / genetics*
Cell Line
Gene Expression Regulation / drug effects
Gene Library
Lipopolysaccharides / pharmacology*
Macrophages / cytology
Macrophages / drug effects*
Mice
MicroRNAs / genetics*
MicroRNAs / metabolism
RAW 264.7 Cells
RNA, Messenger / drug effects
RNA, Messenger / metabolism*
Sequence Analysis, RNA
Toll-Like Receptor 4 / metabolism

Substances

Lipopolysaccharides
MicroRNAs
Mirn155 microRNA, mouse
RNA, Messenger
TLR4 protein, human
Toll-Like Receptor 4
Casp3 protein, mouse
Caspase 3