| microRNAs are a class of small non-coding RNAs, which have recently been reported to play important roles in the regulation of immune responses. However, only limited few studies demonstrated the function of miRNAs in lung epithelial cells and alveolar macrophages in response to Mycobacterium bovis (M. bovis)infection, particularly to the miR-124that its function in lung has not been explored yet. Therefore, it is important to interrogate the mechanism of interaction of miR-124and TLR signaling in lung epithelial cells and alveolar macrophages against mycobacterial infection, which will allows us to better understand the immunoregulatory role of miRNAs in the target cells of mycobacteria.In the present study, we aimed to explore the functional mechanism of miR-124in lung epithelial cells and macrophages in response to mycobacterial infection. To this end, human alverlar epithelial A549cells and murine alveolar macrophage RAW264.7cells were transfected with miR124mimic, miR-124inhibitor or miR-124Nc control, followed by Mycobacterium bovis BCG infection. The key components of TLR signaling pathway and inflammatory factors were first ascertained by ELISA, qRT-PCR and Western blotting assays; the targets of miR-124in TLR signaling cascade were were experimentally validated using luciferase reporte vectors harboring respective3’-UTR of putative target genes; and the in vitro results were further tested in murine models in vivo. These studies thus lay a foundation for further investigating the immunoregulatory roles of miR-124in alverlar epithelial cells and macrophages in response to mycobacterial infection. The main findings of this study are listed as follows:(1) The TLR6, TLR signaling adaptor MyD88, TRAF6, and its down-stream gene TNF-α were predicted as potential target genes of miR-124in TLR signaling pathway, when miRNA tools of miRanda, Target Scan and PicTar were employed. The luciferase reorter assay experimentally validated that miR-124were able to directly bind to and target the3’-UTR sequences of TLR6, MyD88, TRAF6and TNF-α genes;(2) miR-124could negativel regulate the immune responses of alveolar epithelial cells and macrophages in response to BCG infection by directly targeting TLR signaling TLR-6,MyD88, TRAF6, TNF-α, which enable the host cells to avert overwhelming cellular response upon mycobacterial infection.(3) In vitro study demonstrated that overexpression and siRNA-mediated suspression of MyD88expression showed an increased and decreased abundance of miR-124transcripts in cells. This result indicated that the miR-124and TLR signal ing could form a negative feedback loop in alveolar epithelial cells and macrophages during the couse of mycobacterial infection, i.e. an activation of TLR signaling induced miR-124transcription, and an inhibition of TLR signaling activity reduced miR-124expression. Such a mechanism will protect the host cell from the excessive immune response induced by mycobacterial infection.(4) In vivo study further revealed that more abundance of miR-124transcript was found in the peripheral blood lymphocytes of patients with pulmonary tuberculosis, in comparison with that of healthy cohorts. In addition, the transcription levels of miR-124in muring lung tissues and alveolar macrophages in the mice infected with BCG were significantly higher than that of controls; furthermore, the expression of miR-124in the lung and alveolar macrophages of MyD88gene deficient mice was significantly lower than that in wild-type and heterozygous litter mates (p<0.01).Collectively, these results presented in this study demonstrated a negative immunoregulatroy feedback loop of miR-124/TLR in alveolar epithelial cells and macrophages in response to mycobacterial infection. This study herein provides an insight into the underlying mechanism of miRNA in host cells upon mycobacterial infection, which warrants further study for development of novel agents for clinical treatment of Tuberculosis. |
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