| Avian reticuloendotheliosis(RE)is an avian infectious tumorigenic disease caused by avian reticuloendotheliosis virus(REV).REV mainly infects chickens,ducks,geese,turkeys and other avian species,leading to growth retardation,immunosuppression and tumours.Innate immune pathways,as the first barrier against invading pathogenic microorganisms,play an important role in the host antiviral response.The most important pattern recognition receptors(PRRs)in the cytoplasm that recognize viral RNA are the RIG-I like receptors(RLRs).Previously,we used an animal disease model in which the 1-day-old SPF chickens were challenged artificially by REV-SNV,and collected spleen samples at 7 dpi,14 dpi,and 21 dpi for high-throughput sequencing.After integrating analysis of m RNA and miRNA expression profiling data,we found that CASP10 and MAPK10 genes involved in the RLRs receptor signaling pathway were not only significantly differentially expressed,but also significantly negatively regulated by gga-miR-1618-5p and gga-miR-222b-5p,respectively.To further grasp the expression pattern of the above two miRNAs and genes related to the RLRs pathway in REV-SNV infection,we collected tissue samples from previous animal experiments,including bursas of the same days post infection(7 dpi,14 dpi,21 dpi)and spleens of different days post infection(28 dpi,35 dpi,42 dpi),compared to controls,the bursa of Fabricius showed that,at 7 dpi,there was a highly significant down-regulation of gga-miR-222b-5p expression,no significant difference in MAPK10 expression,a highly significant down-regulation of gga-miR-1618-5p expression,and a highly significant up regulation of CASP10 expression,and,at 14 dpi,no significant difference in gga-miR-222b-5p expression,a highly significant up regulation of MAPK10 expression,no significant difference in gga-miR-1618-5p expression,CASP10 expression was highly significantly up-regulated;at 21 dpi,ggamiR-222b-5p expression was significantly down regulated,MAPK10 expression was not significantly different,gga-miR-1618-5p expression was not significantly different,and CASP10 expression was highly up-regulated.However,the results from spleen tissues showed that at 28 dpi,gga-miR-222b-5p expression was extremely upregulated,MAPK10 expression was not significantly different,gga-miR-1618-5p expression was extremely downregulated,and CASP10 expression was extremely upregulated,and at 35 dpi,gga-miR-222b-5p expression was extremely upregulated,MAPK10 was extremely downregulated,gga-miR-1618-5p expression was not significantly different,and CASP10 expression was extremely upregulated.At 42 dpi,there was no significant difference in the expression of gga-miR-222b-5p,MAPK10 and CASP10,while gga-miR-1618-5p was significantly down regulated.Previous high-throughput sequencing results and qPCR validation results showed that MAPK10 and CASP10 might be the potential target genes of gga-miR-222b-5p and gga-miR-1618-5p,respectively.To determine whether they were directly related to each other and to study their targeting relationship,we predicted their binding sites using bioinformatics software targetscan and mirbd,and found that only the 3 ’ terminal non coding region(3’UTR)of MAPK10 and gga-miR-222b-5p had seed binding sites with 3 positions(ugacucg,ugacucgu,gacucgu).Since the first two binding sites were relatively close(654-660,1190-1197)and the third one was far(3890-3896),we constructed two kinds of dual luciferase reporter vectors containing gene fragments of interest respectively,centered on the previous two targets and centered on the third one,and performed dual luciferase reporter gene experiments,which showed that MAPK10 was indeed the target gene of gga-miR-222b-5p.CASP10 acts as an apoptotic factor in the organism,and MAPK10 is a more important tumor regulator in humans and animals.Our study on the regulation of expression of RLRs signaling pathway related miRNAs and potential target genes mentioned above in different immune organs of the same infected day and different infected day of the same immune organ of SPF chicks after REV-SNV infection,we can broaden our understanding of the mechanism of immunosuppression and tumorigenesis caused by REV infection,while contributing to an in-depth understanding of the molecular regulation of miRNA mediated RLRs involved in pattern recognition receptor signaling in chicken immunosuppression. |
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