1. 1. ABSTRACT
MiRNAs, a family of small non-coding RNAs, have emerged as novel post-transcriptional regulators of numerous cellular responses. Although the involvement of miRNAs in the regulation of neuroinflammation in various neurological diseases has been previously studied, their role in the production of inflammatory mediators during microglia activation is poorly understood. In this study, we investigated the role of miR-26a in the modulation of inflammatory response in cultured microglia. We first studied the expression of miR-26a in TLR4 ligand-stimulated primary mouse microglia. We found that the stimulation of TLR4 rapidly reduced the levels of miR-26a in microglia. Overexpression of miR-26a significantly reduced the release of inflammatory cytokines such TNFα and IL-6, whereas knockdown of miR-26a increased the production of these mediators in microglia. Furthermore, using in silico analysis, we found that the activating transcription factor (ATF) 2 is directly targeted by miR-26a. This finding was confirmed by loss and gain of function studies. Similar to the effect of miR-26a overexpression, knockdown of ATF2 inhibited the production of inflammatory cytokines particularly IL-6. Taken together, our results suggest the involvement of miR-26a in regulating microglia mediated production of inflammatory cytokines.
To investigate the expression of miR-26a during microglia activation, primary cultured mouse microglial cells were stimulated with Lipopolysaccharide (LPS) (10 ng/ml) for indicated time points and investigated the expression of miR-26a using real time PCR.
Primary mouse microglial cells were transfected with miR-26a mimic and inhibitor with their respective control using Lipofectamine RNAiMax transfection reagent, to evaluate its effects on release of inflammatory mediators, such as TNFα, IL-6, and PGE2, were assessed by ELISA and EIA, respectively.
Luciferase activity of recombinant vector pmirGlo with mouse ATF2 3′ UTR was measured using the dual-luciferase reporter assay system.
4. METHODS
2. RESULTS
Fig Double immunofluorescence staining for IBA1 (microglial marker) and GFAP (astrocytic marker) in purified primary microglia culture. Most of the cells were IBA1 positive and GFAP negative. Scale bar, 50 μm.
Fig (a) Expression of miR-26a in mouse primary microglia stimulated with LPS for different time points as indicated. (b-c) Mouse primary microglia were transfected with miR-26a mimics or miR-26a inhibitor with their control, as indicated at a final concentration of 50 nM and 200 nM respectively. After 24 h of cell transfection, expression of miR-26a was measured by qRT-PCR. snoRNA55 was used for normalization.
Fig LPS-triggered TNFα, IL-6 and PGE2 release in miR-26a inhibitor transfected microglia. Microglial cells were transfected with miR-26a inhibitor or their respective control inhibitor (Ctrl) at a final concentration of 200 nM. 24 h after the transfection, cells were stimulated with LPS for indicated time. The release of TNFα (a), IL-6 (b) and PGE2 (c) was determined.
Fig Effect of miR-26a mimic on LPS induced p38 MAPK, ERK1/2 MAPK, JNK activation and IκBα degradation in microglia. Cells were transfected with miR-26a mimic (50 nM) or control mimic for 24 h followed by stimulation with LPS for indicated time. Representative blots (upper panel) are shown. (a) p38 MAPK; (b) ERK1/2 MAPK; (c) JNK; (d) IκBα with densitometric analysis (lower panel).
Acknowledgements
Asit Kumar was supported by scholarships as per the State Law on Graduate Funding (LGFG-IGA, Freiburg). The skillful technical assistance of Brigitte Günter is greatly acknowledged. .
Fig (a) Alignment of miR-26a and its target site in the 3′ UTR of ATF2, which was predicted by TargetScan website. BV-2 cells were transfected with miR-26a mimic or miR-26a inhibitor along with controls. The mRNA and protein levels of ATF2 were determined by qRT-PCR (b,c) and western blot (d,e). mRNA and protein levels of control mimic or inhibitor was set to 1. (f) miR-26a binding site on 3' UTR of ATF2 and ligated in pmiR Glo vector. (g) Luciferase reporter activity assay using recombinant vector pmirGlo ligated with mouse ATF2 3′ UTR, was performed after co-transfection with miR-26a mimic (50 nM) and miR-26a inhibitor (200 nM) with their controls in BV-2 cells. After 24 h of cell transfection, firefly luciferase activity was measured and normalized to renilla luciferase activity. The luciferase activity of the control mimic or inhibitor was set to 1.
Fig (a) Primary microglial cells were treated with 10 μM SP for 30 min followed by LPS stimulation for 30 min. Western blot analysis for phospho-ATF2 was performed. (b,c) The release of TNFα and IL-6 in the supernatant was determined following treatment of microglia with 10 μM SP for 30 min followed by LPS stimulation for indicated time points. (d) The mRNA levels of ATF2 were analyzed after 24 h of transfection of control siRNA (siCtrl) or ATF2 siRNA (siATF2) at a final concentration of 100 nM in primary microglia. (e,f) 24 h after transfection of control siRNA or ATF2 siRNA, primary microglia were stimulated with LPS for indicated time points. The release of TNFα and IL-6 in the supernatant was determined.
miR-26a was found differentially expressed and down-regulated in activated microglia.
Overexpression of miR-26a reduced the pro-inflammatory cytokines, whereas inhibition of miR-26a reactivated and enhanced the release of cytokines.
miR-26a directly targets transcription factor ATF2 which negatively regulates the production of IL-6 in activated microglia. However, the exact mechanism by which miR-26a regulating production of TNFα is yet to be explored.
2.1 Culture of primary microglia without astrocyte contamination
2.5 Effect of miR-26a mimic on LPS induced activation of MAPK and degradation of IκBα.
2.3 Effect of miR-26a mimic on release of inflammatory mediators in microglia
2.2 Expression of miR-26a in LPS activated microglia and transfection efficacy of miR-26a mimic and inhibitor
2.7 Effect of JNK inhibitor and siATF2 on LPS induced proinflammatory cytokines production
2.6 Activating transcription factor (ATF) 2 is a direct target of miR-26a
2.4 Effect of miR-26a inhibitor on release of inflammatory mediators in microglia
Fig LPS-triggered TNFα, IL-6 and PGE2 release in miR-26a mimic transfected microglia. Microglial cells were transfected with miR-26a mimics or their respective control mimic (Ctrl) at a final concentration of 50 nM. 24 h after the transfection, cells were stimulated with LPS for indicated time. The release of TNFα (a), IL-6 (b) and PGE2 (c) was determined.
MicroRNA-26a modulates toll like receptor (TLR) 4 induced inflammatory response in microglia
Asit Kumar1, 2, Harsharan Singh Bhatia1, Antonio Carlos Pinheiro de Oliveira3, Bernd L. Fiebich1, 4
1 Department of Psychiatry, Neurochemistry Lab, University of Freiburg Medical School, Hauptstrasse 5, D-79104, Freiburg, Germany
2 University of Freiburg, Faculty of Biology, Schaenzlestrasse 1, D Freiburg, Germany
3 Department of Pharmacology, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, Minas Gerais, Brasil
4 VivaCell Biotechnology GmbH, Ferdinand-Porsche-Str. 5, D-79211, Denzlingen, Germany
3. SUMMARY