The Suppressive Role Of MicroRNA-34a In Macrophages Inflammatory Response And The Underlying Mechanisms

Inflammatory responses are complex events occurring when the host immune system fights against invading pathogens or in the condition of tissue injury, which are mediated by multiple chemokines, cytokines and lipid inflammatory mediators. As the main effector cells in inflammation, macrophages respond quickly and eliminate invading pathogens through production of various inflammatory cytokines. Nevertheless, overproduced cytokines may also cause self damage and result in pathological conditions. Therefore, appropriate control toward macrophage inflammatory response, a double-edged sword, is definitely necessary.MicroRNAs (miRNAs), emerging as a new layer of posttranscriptional gene-regulation mechanism, are small molecules with18to25non-coding nucleotides, which can repress the translation or induce the degradation of their target mRNAs by binding to the3’untranslated region (UTR) of the target genes through imperfect base pairing. MiRNAs have been reported to play critical regulatory roles in various physiological and pathological processes including development, immune response, inflammation and tumorigenesis. While previous studies have identified a number of miRNAs to be functionally involved in the inflammatory response of macrophages, there are still plentiful miRNAs needing to be unraveled for their effects in this process, since the regulatory functions of miRNAs in various biological processes are always completed by networks composed of multiple miRNAs and their targets.MiR-34a is one of the miRNAs with potent tumor suppressive effect, and has been extensively studied in a variety of cancer types. The knowledge that the posttranscriptional regulation of gene expression by microRNAs provides the molecular basis for linking inflammation to cancer indicates a role for miR-34a also in inflammation. However, in contrast to the well-studied and powerful role of miR-34a in tumor, knowledge about its effect on inflammation is almost blank.We pioneer an investigation on the involvement of miR-34a in inflammation by using LPS-stimulated macrophages as an inflammation model, aiming to figure out whether and how this potent tumor suppressor regulates inflammation.PART ONE:MiR-34a negatively regulated macrophage inflammatory responseTo assess the biological function of miR-34a in macrophage inflammatory response, we first examined its expression change in macrophages in response to LPS. Mouse macrophage cell line RAW264.7was stimulated with different doses of LPS for indicated lengths of time and then analyzed for miR-34a expression using real-time RT-PCR. The results showed that miR-34a was downregulated after LPS stimulation. Moreover, the decrease of miR-34a exhibited a dose-dependent manner with LPS treatment. Besides, a decrease of miR-34a expression was also observed in primary peritoneal macrophages and human monocytes THP-1when treated with LPS. These results suggest that LPS could downregulate miR-34a expression in macrophages.Since miR-34a was downregulated after LPS stimulation, we next investigated its role in macrophage inflammatory response. RAW264.7cells were transiently transfected with miR-34a mimics or miR-34a inhibitor to overexpress or knockdown the endogenous level of this miRNA. Cells transfected with miR-34a mimics or miR-34a inhibitor were then analyzed for LPS-induced production of two critical pro-inflammatory cytokines, TNF-α and IL-6, at mRNA level using qRT-PCR and protein level by ELISA assay. As a result, miR-34a mimics induced a significant inhibition in both mRNA and protein expression of TNF-α□and IL-6after LPS exposure compared to the control. MiR-34a inhibitor, in contrast, promoted the LPS-induced production of inflammatory cytokines in macrophages. Therefore, both the gain and loss in function results indicated a strong suppressive role of miR-34a in LPS-induced macrophage inflammatory response.PART TWO:MiR-34a inhibited LPS-induced inflammatory response through directly targeting Notchl in macrophagesGiven the well known mode of miRNAs action, we hypothesized that miR-34a might exert such an anti-inflammatory effect by regulating target genes which promoted LPS-induced inflammatory process. Among the target genes predicted by algorithm of Target Scan and miRanda, Notchl was chosen for further study for its reported important role in macrophage inflammatory response.Notchl was predicted to have a putative miR-34a binding site within its3’UTR, using computational prediction via Target Scan. In order to detect whether miR-34a regulates Notchl through direct3’UTR interaction, the3’UTR of Notchl was cloned into a reporter plasmid downstream from luciferase and reporter assays were performed in RAW264.7cells. As a result, miR-34a obviously repressed the activity of luciferase fused to Notchl3’UTR, which indicated a direct interaction between miR-34a and Notchl in macrophages. To assess whether miR-34a had a functional role in the down-regulation of endogenous Notchl expression, RAW264.7cells were transfected with miR-34a mimics. Expectedly, Notchl expression was decreased by miR-34a mimics at both mRNA and protein level. Besides, realtime RT-PCR analysis for expressions of Notch downstream target genes indicated significantly attenuated Notch signaling activity in LPS-stimulated macrophages after miR-34a overexpression. Taken together, these results showed that miR-34a suppressed Notchl expression and Notch signaling activity in macrophages through direct interaction with Notchl3’ UTR.Aiming to assess whether the regulation of Notchl by miR-34a accounted for its suppressive role in inflammation, experiments of genetic and pharmacological knockdown of Notchl were carried out subsequently. Western blot analysis confirmed that expression of Notchl in RAW264.7cells was effectively inhibited by Notchl siRNA. Real-time RT-PCR and ELISA analysis showed remarkably decreased production of TNF-a and IL-6in LPS treated macrophage after Notchl knock down. Moreover, γ-secretase inhibitor (GSI) DAPT was used to block Notchl signaling, and the efficiency of inhibition was confirmed by western blot analysis. Similar with the siRNA silencing, blockade of Notchl signaling using DAPT reduced the inflammatory response in macrophage after LPS stimulation in terms of reduced production of TNF-α and IL-6. Thus, knockdown of Notchl either genetically or pharmacologically both attenuated macrophage inflammatory response, resembling the suppressive effect of miR-34a overexpression, which indicated that miR-34a might exert its anti-inflammatory role, at least in part, through targeting Notchl. PART THREE:MiR-34a inhibited NF-κB activation during macrophages inflammatory responseNF-κB is a nuclear transcriptional factor that controls the expression of multiple genes implicated in inflammatory response. The effects of Notchl on macrophage inflammatory response have been reported to be mediated by modulating NF-κB activity. Therefore, we next took further investigation upon the effect of miR-34a on NF-κB activation.Luciferase reporter assay, anti-p65immunofluorescence microscopy and western blot were performed to investigate the effect of miR-34a on NF-κB activation. As a result, miR-34a obviously repressed NF-κB luciferase reporter gene activity. The p65nuclear translocation, the degradation of IκBα and the phosphorylation of p65, induced by LPS stimulation, were also decreased by miR-34a overexpression. Taken together, these results showed that miR-34a inhibited NF-κB pathway activation during macrophages inflammatory response.Conclusions:1) MiR-34a negatively regulated macrophage inflammatory response.2) MiR-34a inhibited LPS-induced inflammatory response through directly targeting Notchl and attenuating Notch signaling activity in macrophages.3) MiR-34a inhibited NF-κB activation during macrophages inflammatory response.