| Innate immunity is the body's first line to defense against microbial infection. As an important component of innate immunity, macrophages are central for the innate immune responses to eliminate viral and bacterial infection. Upon recognition of structurally conserved bacterial and viral components termed pathogen-associated molecular pattern (PAMP) through pattern recognition receptors (PRR), macrophages can be activated and secret a large amount of proinflammatory cytokines via a serial of signal transduction. Although activation of macrophages and secretion of proinflammatory cytokines and chemokines are important for the elimination of invading microorganisms, uncontrolled activation may leads to autoimmune and inflammatory diseases. Therefore, it is key of strictly holding macrophage activation and secretion of proinflammatory cytokines and chemokines.microRNAs (miRNAs) are21-22nucleotide(nt), highly conversed, endogenous, small non-coding RNA molecules and play a very important role in the regulation of gene expression. Mature miRNA interacts with a specific mRNA through pairing of nucleotide bases between the miRNA seed sequence and the3'untranslated region (3'UTR) of specific mRNA, and leads to translational repression or degradation of target mRNA. However, the precise functions of microRNA in the regulation of macrophage activation by LPS are not fully understood. Therefore, we investigated the function and the underlying mechanism of microRNA in LPS-induced macrophage activation in this study.Objectives:1. Identifiction of up-or down-regulated microRNAs in macrophage stimulated by LPS;2. Investigating the role of the up-or down-regulated microRNAs in macrophage activation upon LPS stimulation;3. Revealing the mechanism of microRNAs as a negative regulator for LPS-induced expression of proinflmmatory cytokines.Methods:1. Identification and verification of up-or down-regulated microRNAs in LPS-activated macrophage.1.1Confirmation of whether murine macrophages were activated by LPS.After bone marrow derived macrophages (BMDM) were stimulated with LPS for24h, the expression of iNOS, TNF-α and IL-12p40was measured at mRNA level by semi-quantitative PCR to determine the BMDMs were effectively activated.by LPS.1.2microRNA array analysis to identify up-or down-regulated microRNAs.microRNA array analysis was performed with above samples to identify the microRNAs that were up-or down-regulated in activated macrophages by LPS.1.3Veryfication of the microRNAs that were up-or down-regulated with real-time PCR.Validated the data obtained by microRNA array analysis with quantitative real-time PCR.2. Investigating the role of microRNAs in macrophage activation induced by LPS. 2.1After microRNAs mimics were transfected, we detected the expression of proinflammatory cytokines at the level of mRNA and protein.microRNAs mimics were transfected into peritoneal primary macrophages for48h, secretion and expression of proinflammatory cytokines including IL-6and TNF-a was measured by ELISA and real-time PCR after LPS stimulation.2.2After microRNAs inhibitors were transfected, we detected the expression of proinflammatory cytokines at the level of mRNA and protein.microRNAs inhibitor were transfected into peritoneal primary macrophages for48h, secretion and expression of proinflammatory cytokines including IL-6and TNF-a was measured by ELISA and real-time PCR after LPS stimulation.3. Analyzing the mechanism that microRNA affects macrophage activation.3.1After NF-κB-luc reporter plasmid and various adaptors in the LPS/TLR4signaling pathway were transfected together with microRNA mimics, we analyzed the activation of NF-κB.NF-κB-luc reporter plasmid and various signaling molecules in the LPS/TLR4signaling pathway were transfected into HEK293cells together with microRNA mimics, then we determined the function of microRNA on the NF-κB activation in the LPS-induced NF-κB activation by luciferase reporters.3.2Identifying the potential microRNA targets in the LPS/TLR4signaling.The microRNA prediction programs including miRWalk, miRanda, RNA22and Targetscan were applied to analyze the potential microRNA targets. The3'UTR of corresponding targets was cloned and fused downstream of the firefly luciferase gene to generate a reporter construct (p50-3'UTR-WT and p50-3'UTR-MU), then transfect p50-3'UTR-WT/p50-3'UTR-MU and microRNA into HEK293cells and detect luciferase activity.3.3Evaluating the role of microRNA for the expression of endogenous target protein through Western blotting.microRNAs mimics were transfected into peritoneal primary macrophages for48h, expression of target protein was measured by Wsetern blotting.3.4Analyzing the mechanism that microRNA affects macrophage activation. microRNAs mimics were transfected into peritoneal primary macrophages for48h, the binding between target protein and the promoter of proinflammatory cytokine was analyzed by CHIP after LPS stimulation for1h.Results:1. miR-210was induced in macrophages activation by LPS.1.1Proinflammatory cytokines were remarkably up-regulated24h after LPS stimulation.Compared with un-stimulation, proinflammatory cytokines, such as iNOS, TNF-a and IL-12p40, were significantly increased after LPS stimulation for24h. And above results indicated LPS effectively activated these murine macrophages.1.2microRNA array analysis was performed and revealed that the expression of several miRNAs was either up-regulated or down-regulated upon LPS challenge.We performed microRNA array with above verified samples and found miR-155, miR-147, miR-146were induced～344,18and4fold, respectively, which have been previously shown to be induced in LPS stimulated macrophages. In this report, miR-210, which were induced about6fold, was chosen for further studies.1.3Consistent with microRNA array analysis, miR-210was also up-regulated by real-time PCR.We synthesized specific primers for miR-210and analyzed the expression of miR-210with real-time PCR. Compared with control, miR-210was induced approximately8fold after LPS stimulation for24h.2. miR-210negatively regulated macrophage activation by LPS.2.1miR-210mimics inhibited expression and secretion of proinflammatory cytokines.Expression of miR-210was validated by quantitative real-time PCR after transfection of miR-210mimics, and we found miR-210were up-regulated about 800fold. Then we demonstrated LPS-induced secretion of proinflammatory cytokines including IL-6and TNF-a was significantly decreased in miR-210mimics transfected macrophages.2.2miR-210inhibitor enhanced expression and secretion of proinflammatory cytokines.Transfection of miR-210inhibitors greatly attenuated miR-210expression in macrophages compared with control miRNA inhibitors. As well as, inhibition of miR-210expression increased LPS-induced IL-6and TNF-a secretion.3. miR-210played a negatively regulatory role by targeting3'-UTR of NF-κB1.3.1miR-210suppresses NF-κB activation.After NF-κB-luc reporter plasmid and various signaling molecules in the LPS/TLR4signaling pathway were transfected into HEK293T cells together with miR-210mimics, MyD88, TRAF6, TAK1plus TAB1, and IKKβ induced NF-κB luciferase activity was greatly inhibited by miR-210mimics transfection, compared with control microRNA mimics transfection. These data indicate that miR-210targets a molecule downstream of IKKβ to inhibit NF-κB activation and subsequent production of proinflammatory cytokines.3.2miR-210interacted with3'UTR of NF-κB1and inhibited its luciferase actitivity.The microRNA prediction programs including miRWalk, miRanda, RNA22and Targetscan were applied and a putative miR-210binding site was predicted in the3'UTR region of NF-κB1with these four programs. Therefore,3'UTR of p105(WT and two mutants) was cloned and fused downstream of the firefly luciferase gene to generate a reporter construct. Transfection of miR-210mimics significantly inhibited the luciferase activity of the report construct with the wild type3'UTR. However, after the miR-210binding site in the3'UTR of p105was mutated, the inhibitory effect of miR-210on the luciferase activity was abrogated.3.3miR-210inhibits endogenous NF-κB1expression.Based on above results, we analyzed the expression of endogenous p105and p50by Western blotting and found they were greatly decreased with transfection of miR-210mimics, compared with that of control microRNA mimics transfction.3.4miR-210inhibits p50/p65binding to IL-6promoter.CHIP experiments were performed to investigate the p65/p50binding to the IL-6promoter, and we found that transfection of miR-210mimics greatly attenuated LPS-induced p50binding to the NF-κB site in IL-6promoter.Conclusion:1. LPS stimulation led to the increased expression of miR-210in murine macrophages;2. miR-210inhibits macrophage activation induced by LPS;3. miR-210inhibits the expression of p50via interacting with its3'-UTR and subsequent p50binding to IL-6promoter and expression of proinflammatory cytokines.Innovation and significances:1. In this study, it is the first time that we demonstrated as follows:1) miR-210as a negative regulator to inhibit LPS-induced production of proinflammatory cytokines;2) miR-210targets NF-κB1by interacting with the3'UTR of NF-κB1;3) p50binding to IL-6promoter were greatly decreased by transfection of miR-210mimics.2. Our studies provided new experimental evidence for negative regulation of macrophage activation, and may provide novel idea for diagnosis and therapy of macrophage-mediated inflammatory diseases..
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