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 LPSactivated 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 LPSinduced 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.

Identification of RNA-binding Proteins in Macrophages by Interactome Capture

“…Interestingly, we discovered that LPS-induction of RAW 264.7 macrophages leads to miR-155-mediated CASPASE-3 downregulation and decelerated apoptosis, thereby sustaining macrophage activity (87). This is in agreement with a role of P23 in preventing ER-stress-induced apoptosis (88,89).…”

miR-155 Regulated Inflammation Response by the SOCS1-STAT3-PDCD4 Axis in Atherogenesis

“…Several studies showed that miR-155 regulated the production of inflammation mediator via direct target [36–38]. In our study, HEK293 cells were cotransfected with the wild-type (WT) or mutated (Mut) SOCS1 luciferase reporter vector, together with miR-155 mimic or miR-NC, for 24 h. Luciferase activity was significantly inhibited in cells transfected with WT SOCS1 and miR-155 mimic, but not in cells transfected with mutation SOCS1 and miR-155 mimic (Figures 5(a) and 5(b)).…”

MicroRNA-155 Participates in Smoke-Inhalation-Induced Acute Lung Injury through Inhibition of SOCS-1

“…Additionally, it is reported that upregulated miR-155 also exhibits suppressive effects on apoptosis of specific activated inflammatory cells to maintain their critical functions in immune response [27]. MiR-155 also prevents apoptosis in macrophages induced by LPS [28]. Although this phenomenon might be helpful for the host defense against the invasion of pathogens, prolonged life span of inflammatory cells might contribute to excessive inflammatory responses which cause tissue injury.…”