The Regulatory Mechanism Of "MiR-155-IRF2BP2-Interferon" Axis Of Monocytes When Confronting Cryptococcus Neoformans

background and ObjectiveBrain infection caused by Cryptococcus neoformans can cause inflammation of the meninges and brain parenchyma, one of the common manifestations of meningoencephalitis. One million people are estimated to suffer cryptococcal meningitis globally,of which more than 60% die within 3 months of diagnosis. Most people contract infection by inhalation of spores or yeast cells causing the infection, then the invasion colonize to the lung tissue. In the lungs,there are a large number of alveolar macrophages to clear Cryptococci, but some are not be cleared,cause pulmonary infection and/or disseminate to other body organs. Interestingly, Cryptococci survive and multiple inside phagocytes, through the "Trojan horse" to penetrate the blood-brain barrier, and ultimately caused cryptococcal meningitis, however, the mechanism of attracting C. neoformans colonization to the brain are not yet known.In recent years, small RNA (microRNA, miRNA, miR) regulation of gene expression as a "star element", play a key role in ontogeny, immune response, cell proliferation, differentiation, apoptosis, migration, angiogenesis and tumor formation. miRNAs are a class of single-stranded of about 20-25 non-protein-coding small RNA nucleotides, is a key negative regulator in the post-transcriptional gene expression. miRNA targeted mRNA can be combined with the 3’untranslated region, the 5’ untranslated region or the coding region, the translation is hindered or degradation of the targeted mRNA, thus inhibiting the translation of post-transcriptional gene. The function of miRNA in many fields were expanded study, which miR-155 as a member of miRNA family, significantly in the immune system,involved in T cells, B cells and macrophages, its expression is often induced by specific antigens、IFN-γ etc. Studies have found that miR-155 knockout mice showed a severe immune dysfunction, including B cells, T cells and antigen presenting cells,in addition, there are many results about the miR-155 signaling and regulation mechanisms of type I interferons in antiviral innate immunity.However, miR-155 in monocytes stimulation of Cryptococcus neoformans regulating type I interferon have not been studied. This research provides a theoretical and experimental basis for finding an effective target for treatment of cryptococcosis.This study aims to analyze the miR-155 in heat-killed cryptococcal stimulated monocytes induced changes of type I interferon, to clarify its target genes, explore the possible mechanism of miR-155 regulate the immune response of monocytes.Methods1, By density gradient centrifugation from healthy controls and patients with cryptococcosis in peripheral blood mononuclear cells, MACS isolated monocytes, use Trizol to extract RNA, reverse transcription cDNA, quantitative real-time polymerase chain reaction to detect the expression levels of miRNA.2, By Real-time PCR to verify the results of microarray,detect the expression of IFN-α and IFN-γ monocyte cell line stimulated by heat-killed cryptococcal; Lipofectamine 2000 liposome-mediated transfection, the miRNA-155 mimics and miRNA-155 inhibitor transfected into monocyte cell line U937 and use Real-time PCR to detect the expression of miRNA-155 and interferon transfection.3, By use of the forecasting software TargetScan, PicTar and miRBase to predicted the targets of miR-155. Then use dual luciferase reporter gene technology to verify whether 3’-UTR of a target gene have a direct inhibitory effect.4.By use of siRNA to silence the expression of IRF2BP2. Use Real-time PCR and Western blot to detect after silence IRF2BP2 the changes of IFN-α and IFN-γ.Results1.Compared with the healthy control group, the level of interferon significantly decreased in patients with cryptococcosis.However,miR-155 expression was significantly increased in monocytes line.2.After stimulation with Heat-killed Cryptococcus neoformans, level of IFN-α and IFN-γ in monocyte cell line increased, using "gain-and loss-of-function" found that miR-155 may negatively regulates IFN-α and IFN-γ.3, Using a dual luciferase reporter gene technology, Real-time PCR and western blot validation of miR-155 and IRF2BP2 3’UTR has targeted relationship.4, Using siRNA silent the expression of IRF2BP2, the ability of U937 induce IFN-α and IFN-γ decreased after stimulated with heat-killed Cryptococcus neoformans.ConclusionExpression of miR-155 in monocytes of Cryptococcosis patients increased, IRF2BP2 is a target of miR-155. In monocytes, we concluded that miR-155 by target IRF2BP2 negatively regulates IFN-α and IFN-γ production, thereby inhibiting monocytes antifungal response, causing cryptococcal infection disease. Therefore, miR-155 may be a potential therapeutic target of immunotherapy cryptococcosis.