| The innate immunity is the first line to defense against invading pathogens and is also essential for the subsequent adaptive immune response. The interferon signaling represents the key aspect of the anti-viral immunity. Thus, the studies focused on the type I interferon have been the hot issues in both virology and immunology fields. As one of the post-transcriptional gene regulatory mechanism, miRNA was involved in a wide range of life processes which include development, neuron development, apoptosis, proliferation, fat metabolism and hormone secretion. In the recent years, growing evidences have demonstrated that miRNA plays a negative regulatory role in the innate immunity. The present study tries to discover novel miRNAs who regulate the expression of several key signal molecules in the anti-viral signaling. With this effort, we try to deepen the understanding of the miRNA based negative regulatory mechanism. This study will provide theoretical basis for the future miRNA study and the development of miRNA as a potential target for disease diagnosis and therapy. The detailed research includes the following aspects:1. Screening for dsDNA-induced differently expressed miRNA and function study.dsRNA, dsDNA, LPS and flagellin were recognized by the cell surface PRRs and trigger the cellular innate immunity. LPS and dsRNA induced differential miRNA expression have been well characterized. As the bypass products of viral replication in the host cells, dsDNA induced differently expressed miRNA remains largely unknown. Thus, we use miRNA microarray to screen for the differently expressed miRNAs in response to the dsDNA mimic poly(dAT:dAT) stimulation. We found ten up-rugulated miRNAs which include miR-518b, miR-492, miR-181a and miR-331-3p; five down-regulated miRNA which include miR-383, miR-618and let-7a. We utilized the miRNA specific Real-Time PCR to validate the result. Furthermore, we predicted miRNAs'targets and made validation by using luciferase repoter system. We found that miR-181a negatively regulate the interferon signaling by targeting receptor DAI.2. Screening for the miRNA who regulate IL-6expression and mechanism studyInterleukin-6(IL-6) is a pleiotropic cytokine. Upon inflammatory stimulation, the IL-6expression was highly induced and promotes other pro-inflammatory cytokines and chemokines secretion. The induced IL-6expression will intensify the inflammation to eliminate the pathogens rapidly. However, the uncontrolled IL-6expression is harmful to body health. The sustained inflammation plays an important role in the carcinogenesis. Thus, the negative regulatory mechanism to make sure the appropriate IL-6expression is indispensable to the body health. We used a bioinformatics technique to predict miRNA targeting IL-6. We found miR-365negatively regulate IL-6expression by using luciferase gene reporter system. Mutagenesis analysis revealed the precise binding site for miR-365in TBK-1's3'UTR region. ELISA assay demonstrated that miR-365negatively regulate IL-6protein expression. Real-Time PCR showed that miR-365repress the expresson of IL-6without decreasing its mRNA stability. Analysis of the miR-365promoter showed that two transcription factors, Spl and NF-κB, cooperatively regulate the transcription of miR-365. We also demonstrate that the MAPK/ERK pathway contributes to the regulation of miR-365by using specific inhibitor. Furthermore, after analysis of potential targets of miR-365, we found that it is possible that miR-365can also regulate IL-6exprssion indirectly.3. Screening for the miRNA who regulate TBK-1expression and mechanism studyTBK-1is the key signal molecule in the type I interferon signaling pathway. Its primary function is the phosphorylation of transcription factor IRF3and IRF7which lead their translocation into nucleus and the initiation of IFN-β transcription. TLR-and RIG-I-dependent anti-viral signaling converges at TBK-1-IRF3model emphasizes TBK-1's essential role in the anti-viral immunity. In order to discover potential miRNA which negatively regulate interferon signaling by targeting TBK-1, we predicted miRNAs which target TBK-1mRNA. We found miR-155out of the candidates inhibited reporter gene's expression. Ectopic expression of miR-155significantly repressed TBK-1protein expression. In contrast, miR-155inhibitor increased TBK-1protein expression. Both of the results demonstrated that miR-155negatively regulate TBK-1expression. Mutagenesis analysis revealed the precise binding site for miR-155in the TBK-1's3'UTR region. Furthermore, ectopic expression of miR-155decreased IRF3and NF-κB activity, which in turn blocked IFN-β transcription. TBK-1knockdown and gain-of function assay demonstrated that miR-155's inhibitory effect is achieved through blocking TBK-1expression.In order to evaluate the effect of miR-155on poly(dAT:dAT) induced interferon expression, we determined the miR-155's effect on VSV or SEV replication. We found that ectopic expression of miR-155promoted VSV and SEV replication in HEK-293by blocking interferon secretion. We also found that miR-155was induced by inflammatory stimulation. NF-κB and MAPK signaling are highly involved in the transcriptional regulation of miR-155. Our data showed that the inducible expression of miR-155may represent an important negative feedback regulation of interferon signaling pathway.4. Screening for the miRNA who regulate JAK-1expression and mechanism studyJAK-STAT is one of the most important signaling which links interferon and interferon stimulating genes. JAK-1is a member of Janus non-receptor tyrosine kinas family. When interferons binding to its receptor in the cell surface. JAK-1phosphorylates STAT-1and STAT-2, two members of STAT family. Subsequently, they translocate from cytoplasm into nucleus and initiate the transcription of interferon stimulation genes. JAK-1also plays an important role in the cytokine signaling which include IL-2, IL-4, IL-6, IL-10and IL-13. Untill now, no miRNA was indentified as the regulator of JAK-1. In order to discover miRNA which target JAK-1, we screened and identified miR-23a as a novel regulator of JAK-1gene by binding to its3'UTR. Site-direct mutagenesis assay identified the precise binding region for miR-23a. Western-blot assay indicated that miR-23a could significantly repress the JAK-1protein expression and this is mainly through degrading JAK-1mRNA. Luciferase reporter gene based assay and Real-Time PCR result showed that miR-23a could inhibit interferon induced ISRE activity and interferon-stimulating gene expression. Our data also showed that miR-23a could be induced in the late phase after the immuno-stimulation. Pharmacological inhibitor assay showed that NF-κB and MAPK/JNK is involved in the transcriptional regulation of miR-23a. Our study indicated that the inducible miR-23a in response to interferon provides a negative feedback mechanism to keep the proper activity of JAK-STAT signaling.
|
PDF Full Text Request