| The immune response to viral infection mechanism in organism is a hotspot inimmune circles.After virus infection, resistance would occur, virus might struggle toescape immune response process. Mchanisms for understanding and mastering struggle toeffectively control the virus infection is of great significance. In the process of viralinfection, immune cells can identify viral components through pattern recognitionreceptors, thereby activating downstream signaling pathway to express type I interferon(IFN) and pro-inflammatory cytokines. As an important class of cytokine, type I interferonhas a wide range of physiological activities, including anticipating in the immuneresponses of anti-virus, anti-tumor and others, and also performing the immune regulationfunction. Also, the viruses achieve immune escape and promote their own expression invivo by acting on the type I interferon signaling pathway frequently.Moreover, type Iinterferon has been widely used in the immune treatment of clinical diseases. However,according to the epidemiological analysis, the effects of type I interferon variessignificantly in the treatments of different types of infection. Understanding the expressionof interferon and its regulatory mechanism of downstream signal, has important scientificsignificanceCurrently the study of the type I interferon signaling pathway has reached aconsiderable depth. The view that the type I interferon performs signaling cascadeconduction through the JAK/STAT pathway has been confirmed by many studies; thefunctions of regulation signaling molecules such as SOCS family on the pathway of type Iinterferon have also been gradually revealed. However, the research on whether type Iinterferon signaling pathway is regulated by microRNA (miRNA) remains less.Micro-RNAs (miRNA) is a class of highly conserved non-coding RNA fragmentswith rich expression and a general length of18to25nt within cells. miRNAs exhibit a brand new mechanism of post-transcriptional gene expression regulation. It inhibits geneexpression by binding the3’UTR region of mRNA of the target gene. The cover of the firstissue of Cell in2010was reserved for the research of transcriptional regulation of miRNAsto gene expression, which indicated that researches on miRNA have become a hot topic inthe fields of life science research. Yet only a fraction of the biological functions of miRNAget articulated. These miRNAs are responsible for the regulations of cell growth and tissuedifferentiation thus highly relevant to the development and diseases during life course, andplay an important role in a variety of physiological and pathological processes includingthe inflammatory responses and tumorigenesis.Based on the above-mentioned backgrounds, through high-throughput miRNAmicroarray screening, we discovered that in the process of infection to the mouseperitoneal macrophages by VSV (vesicular stomatitis virus), intracellular expression ofmiR-93was significantly reduced, thereby the negative regulation to JAK/STAT pathwaywas reduced and the virus replication was suppressed.Part I: miR-93down-regulation caused by induced macrophages after RNAviral infection depends on the triple-methylation occurred at locus H3K4and locusH3K27caused by RIG-I/JNK pathway, rather than TLR/Myd88pathway.First, we have confirmed the down-regulation of miR-93after RNA virus infectionthrough RT-PCR experiments, and studied the mechanisms of the down-regulation ofmiR-93after RNA virus infection. It was discovered through experiments that after theVSV or SeV infection, miR-93emerged down-regulation expression in macrophages.Meanwhile, we dignosed the miR-93expression in PBMC of Flu-A patients, the resultindicated that miR-93was down-regulated under Flu-A virus stress. Futhermore, thedown-regulation trend of miR-93expression after VSV infection in peritonealmacrophages of TLR3, TLR4, TLR9and Myd88-deficient mice did not change. Thisexcluded the influence of TLR signal on the expression of miR-93. While inRIG-I-deficient mice, miR-93did not show down-regulation after virus infection. So it canbe regarded that the down-regulation of miR-93in viral infection is dependent on RIG-I.As a control, the down-regulation of LPS-induced miR-93had no influence on theRIG-I-deficient macrophages. Further, by adding pathway inhibitors to the VSVstimulation system of macrophages, we discovered that the down-regulation of miR-93is dependent on the JNK pathway. Further again, the results of ChIP indicated that thedown-regulation of the miR-93is related to the methylation at locus H3K4anddemethylation at locus H3K27of histones at the promoter region of miR-93.Part II: The down-regulation expression of VSV-induced miR-93is capable ofinhibiting viral replication through enhancing the downstream signaling of type Iinterferon.In order to study the biological significance of miR-93down-regulation after RNAvirus infection further, we examined the influences of miR-93on VSV replication inmacrophages. The results indicated that after the high expression of miR-93, VSVreplication within cells was enhanced, while the amount of virus in the supernatant wasalso significantly increased.For purpose of exploring the mechanism of this phenomenon, we first studiedwhether the up-regulation expression of miR-93could cause any influence on theproduction of type I interferon itself. The results of miRNA and protein levels indicatedthat the miR-93mimics and inhibitor did not influence the production of VSV-inducedcytokines like IFN-β, TNF-α, Il-6, etc. In the meantime, phosphorylation degree of IRF3,NF-κB, P38, ERK, and JNK remained the same. So we focused on the downstream signalvariation of the type I interferon. Existing research results indicate that the VSV virusinfections can cause the phosphorylation of STAT1molecules, and reach a peak after24hours. The results from Western indicate that the phosphorylation level of STAT1wassuppressed after the high expression of miR-93. Apply IFN-β to stimulate the macrophageswith highly expressed miR-93, and discovered that the expression of the downstream genesat the pathway of type I interferon-ISG15and IP10-were also suppressed. These resultssuggest that miR-93promoted viral replication by inhibiting downstream signaling of typeI interferon pathway, and it has no influence on the production of interferon itself.Part III: miR-93inhibits the signal transmission of type I interferons andpromote virus replication by targeting JAK1protein.In the following part, we are about to discussed the target molecules on whichmiR-93influenced to inhibit the JAK/STAT pathway and its downstream signaling.Through the TargetScan (www.targetscan.org) online software, we discovered conservative target sites of miR-93at861-867and1085-1091regions at3’UTR region ofJAK1. While the JAK1protein are the critical signaling molecules of JAK/STAT signaltransduction pathway, so we speculated that it was the down-regulation of miR-93after theVSV stimulation to macrophages that promoted the translation of JAK1protein. So wehave confirmed that both of the mRNA level and protein level of JAK1protein weredecreased after the high expression of miR-93in macrophages through RT-PCR andWestern Blot experiment. And the double fluorescence report results also proved thatmiR-93may target the3’UTR region of JAK1and function.To further demonstrate that the target site of miR-93to type I interferon pathway isJAK1, we conducted siRNA and high expression experiment of JAK1. We discovered thatthe siRNA of JAK1had a similar function to the highly expressed miR-93, that is to inhibitthe replication of VSV virus. The up-regulation expression of MiR-93inhibitor to theIFN-β-induced ISG15can also be blocked by siRNA of JAK1. Build the RAW264.7stabletransfected cell lines with highly expressed JAK1(without3’UTR) further, after VSVstimulation, miR-93cannot perform antivirus activity through regulating JAK/STATpathway and its downstream.Part IV: Study of miR-93in vivo effectTo further validate the in vivo effects of miR-93, we synthesizedcholesterol-modified miR-93in vivo transport reagents-Agomir-93(Guangzhou RiboBioCo., Ltd.), and stimulate high in vivo expression of miR-93in mouse through tail veininjection. The experiment confirmed that in VSV infection after in vivo high expression ofmiR-93, the JAK1protein expression in liver, lung and spleen decreased; HE staining oflung frozen section showed that the number of nucleated cells in lungs increasedsignificantly after the high expression of miR-93, which indicated that the viral replicationhas been enhanced; the viral loads in liver and spleen were also improved after the highexpression of miR-93; Compare the mice of high miR-93expression group with thecontrol group, the amount of IFN-β in serum did not change. The above-mentioned in vivotests demonstrated from another aspect that the down-regulation of miR-93virus infectioncontributed to downstream molecules’ antivirus effects of type I interferon.In summary, we consider that the miR-93is capable of promoting the expression of JAK1molecules through down-regulation after infection, and up-regulating thedownstream genes of type I interferon so as to perform antivirus effects. |
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