The Role And Mechanism Of MicroRNA-155in Anti-Inflammatory Effect Of Glucocorticoids

Because of the recognition of their anti-inflammatory effects, glucocorticoids (GCs) have remained at the forefront of therapies to control inflammatory and autoimmune diseases for the past decades. Natural and synthetic GCs are effective for dampening the inflammatory response against pathogen invasion and protecting the host from excessive inflammation. Thus, they are widely used to treat both acute and chronic inflammation, such as severe shock, asthma, inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. However, their clinical efficacy is compromised by the metabolic effects of long-term treatment and resistance. For the efforts to be effective, greater insights are required into the mechanisms by which GCs exert their anti-inflammatory and immunosuppressive actions.One possible mechanism whereby GCs mediate their effects is through Glucocorticoid receptor (GR), a ligand-activated transcription factor of the nuclear receptor super-family. GR-GCs complex may bind to specific DNA response elements in the promoter region or interact with other transcription factors, and thereby directly or indirectly regulate the transcription of target genes. A large number of protein-encoding genes regulated by GCs have been identified in immune inflammatory response, especially some pro-inflammatory molecules downstream of TLR signaling. However, knowledge of non-coding RNAs, for example, microRNA (miRNA) remains largely unknown.Recently, a novel family of non-coding RNAs, referred to as miRNAs, provide additional controls to the complicated regulation of gene expression. They are produced from actual genes within the genomic loci of cells and then processed by Drosha and Dicer into mature miRNAs. Mature miRNAs complementarily bind to the3’untranslated region of the mRNA transcripts of their targeted genes with the seed sequences, leading to either mRNA degradation or translational repression. Increasing evidences have indicated miRNAs to be key regulators in various biological processes, including innate and adaptive immune responses. Indeed, a number of miRNAs, such as miR-146a, miR-147, let-7e, let-7i and miR-155, were shown to become induced in response to TLR4signaling and functionally promoted or inhibited inflammation. Despite advances in our understanding of miRNAs biology, little is known about the regulation of miRNAs by GCs in inflammatory process and their roles in the anti-inflammatory effect of GCs.In this study, we identified several miRNAs that are regulated by GCs in LPS-induced macrophage inflammatory response. One of them, miR-155, was further shown to be downregulated by GCs, and its downregulation was critical for the anti-inflammatory effect of GCs. Besides, GCs decreased the miR-155expression in GR and NF-KB-dependent manners. The NF-κB binding site located in the promoter region of BIC was important for mediating the GCs-driven suppression of miR-155in response to LPS stimulation. Together, these results suggested miR-155to be a novel target through which GCs inhibit LPS-induced macrophage inflammatory response.Part Ⅰ GCs regulate miRNA expression profile in inflammationMacrophages are important innate immune cells that are crucial for inflammatory responses. The infiltration of the inflammatory cells like macrophages is an important process of inflammation, of which macrophages have two-edged functions. On one hand, the activation of macrophages produces inflammatory cytockines to enhance the immune response and protect the host against the invasion of pathogens. On the other hand, excessive or prolonged activation of macropages will lead to tissue injury and contribute to pathogenesis. Raw264.7cells are murine monocyte-macrophages which mimic the inflammatory reaction of primary cells following the stimulation of LPS, and thereby allowing it to be a recognized cellular model in inflammation research. Therefore, this inflammation model was used in the present study.Raw264.7cells were pretreated with dexamethasone (Dex) or vehicle control for1h and then stimulated with LPS for24h. Dex significantly reduced the expression of TNF-α, IL-6, and iNOS mRNAs and the protein levels of TNF-α, IL-6, and NO. These results indicate that GCs inhibit LPS-induced macrophages inflammatory response.In an effort to determine whether GCs could modulate any miRNAs during this process, we performed a miRNA screening. RNA was then extracted, and differentially expressed miRNAs were screened using a microRNA array analysis.We used a1.5-fold change threshold and a P-value cutoff of0.05to identify miRNAs differentially expressed between Dex-treated and untreated RAW264.7cells. Ninety-six miRNAs were upregulated and30miRNAs were downregulated significantly after stimulation with LPS, whereas60miRNAs were upregulated and137miRNAs were downregulated significantly when LPS was combined with Dex treatment. Several miRNAs that are known to be induced downstream of TLR4signaling (let-7i, let-7e, miR-146a, miR-147, and miR-155) and significantly downregulated miRNAs (miR-26b, miR-148, miR-32b, and miR-301b) were further validated by real-time RT-PCR. Dex profoundly inhibited the expression of miR-146a, miR-147, miR-26b, miR-148, miR-32b, miR-155, and miR-301b, but had no significant influence on let-7i and let-7e.These results demonstrate that GCs not only regulate the expressions of protein-coding genes but also none-coding genes like microRNA.Part Ⅱ Glucocorticoids inhibit inflammatory response by downregulating microRNA-155To assess whether miR-146a, miR-147, miR-26b, miR-148, miR-32b, miR-155, and miR-301b have functional roles in GC-modulated macrophage inflammatory response, Raw264.7cells were transiently transfected with mature miRNA mimics. Cells were then stimulated with LPS along with Dex or vehicle control.Compared with miR-NC, miR-155significantly increased the release of the proinflammatory molecules TNF-α, IL-6, and NO in GC-treated Raw264.7cells. These results indicate that ectopic expression of miR-155reverses the anti-inflammatory effect of GCs on LPS-induced macrophage inflammatory response.Because GCs could decrease miR-155expression, we wanted to investigate further the influence of such an alteration on the LPS-induced macrophage inflam-matory response. Knockdown of miR-155expression by its inhibitor significantly decreased the production of TNF-α, IL-6, and NO in LPS-induced Raw264.7cells, which was consistent with the effect of GCs. These results provide further evidence that inhibition of miR-155may be involved in the anti-inflammatory effect of GCs on LPS-induced macrophage inflammatory response.Mature miRNAs complementarily bind to the3’untranslated region of the mRNA transcripts of their targeted genes, containing seed sequences, leading to translational repression. In order to investigate the molecular mechanism through which miR-155regulate the effect of GCs, we performed bioinformatics’analysis to search for miR-155target genes. Three prediction algorithms (PicTar, Target Scan and miRanda) were used to identify putative target genes and eighty-three candidate genes were commonly predicted to be the possible targets of miR-155. Among them, SOCS1functioning as a negative mediator in inflammation was selected for further analysis. The relative luciferase activity of reporter containing the potential binding site was reduced by miR-155in293T cells. Significant decreases in SCOS1protein levels were also detected in both miR-155-transfected Raw264.7cells. These results indicate that SCOS1is a direct target of miR-155.We next examined the influence of GCs on SOCS1expression. Raw264.7cells were pre-treated with GCs and then stimulated with LPS. The protein levels of SOCS1were measured by western blot. We found that the expression of SOCS1was significantly increased following the treatment of GCs compared with control.All these findings strongly imply miR-155as a key regulator in the anti-inflammatory effect of GCs, indicating that GCs exert their function, at least in part, through the downregulation of miR-155and the upregulation of its target SOCS1.Part III The mechanism of GCs-mediated miR-155expressionWe further examined the effect of GCs on miR-155expression in murine primary macrophage and human monocyte cell line THP-1. Consistent with the results in Raw264.7cells, LPS greatly induced miR-155expression, while Dex reduced its expression both in murine primary macrophage and human monocyte cell line THP-1. In addition, we explored the influence of GCs on miR-155expression in vivo. MiR-155expressions in liver and spleen were also downregulated by Dex compared with the control group. These results suggest that GCs inhibit miR-155expression in response to LPS stimulation in both murine and human monocyte cells, as well as in mouse in vivo.GR is expressed in most of the cells and regulates genes controlling the development, metabolism, and immune response. To directly examine whether the effects of Dex on miR-155were under the control of GR, RU486was used to block the GR-mediated effects and then treated with Dex and LPS. The inhibition of Dex on miR-155expression induced by LPS was reversed following the treatment of RU486. Therefore, it indicates that the inhibitory action of GCs on miR-155expression in response to LPS stimulation is dependent on GR activation.Stimulation of TLR4by LPS triggers the activation of multiple signaling pathways, most importantly including MAPKs and NF-κB pathways.In order to investigate the molecular mechanism involved in GCs-mediated miR-155expression, we first examined the influence of GCs on MAPKs and NF-κB activation induced by LPS.We found that pretreatment with Dex suppressed p38MAPK phosphorylation compared with the control group during both phases. In contrast to p38MAPK, phosphorylations of ERK and JNK were not significantly altered by Dex treatment. LPS-induced p65nuclear translocation was also suppressed in Dex-treated cells.We next examined whether p38MAPK and NF-κB pathways regulate the LPS-induced expression of miR-155by using p38kinase inhibitor SB203580or NF-κB inhibitor BAY-117082. Raw264.7cells were pretreated with the inhibitor and then stimulated with LPS. However, inhibition of p38kinase by SB203580did not exhibit a significant effect on the induction of miR-155by LPS. However, inhibition of NF-κB pathway by BAY-117082significantly reduced miR-155expression in LPS-treated Raw264.7cells, reflecting the importance of NF-κB pathway in the induction of miR-155by LPS. Therefore, these results indicate that GCs inhibit miR-155expression in response to LPS stimulation through suppression of NF-κB activation rather than MAPK pathway.We next examined whether GCs could inhibit transcription of miR-155. Raw264.7cells were pretreated with Dex and then stimulated with LPS for12h. Both pri-miR-155and pre-miR-155were rapidly induced in response to LPS, and significantly reduced when pre-treated with Dex. Therefore, GCs inhibited the expression of both pri-transcript and precursor of miR-155, suggesting that GCs may act upstream to inhibit the actual transcription of the host gene of miR-155. To gain insights into possible conserved regulatory elements in the BIC promoter, the BIC promoter (5000bp upstream to500bp downstream) was then analyzed for potential transcription factor binding sites using the program TFSEARCH. However, no GRE was found within this region. Instead, AP-1, c-Ets and NF-κB site with high probability scores were identified. Dex was no longer able to inhibit luciferase activity when the NF-κB site was mutated. These results indicate that NF-κB pathway and NF-κB binding site are important in mediating the GCs-driven suppression of miR-155expression in response to LPS stimulation.In summary, the present study for the first time systematically scanned the miRNA expression profile regulated by GCs in LPS-induced macrophage inflammatory response. Identification of miR-155as a novel target of GCs may explain, at least in part, the molecular mechanism of the anti-inflammatory effect of GCs on LPS-induced macrophage inflammatory response. GCs inhibit miR-155expression through GR and NF-κB dependent manners.Such inhibition of miR-155expression results in the expression increase of its target SOCS1, and thereby leading to the suppression of inflammatory response.This study therefore sheds light on a new role for miRNA in mediating suppressive effect of GCs in inflammation, and may provide a basic rationale for new approaches in effort to develop anti-inflammatory therapeutics.