Study On Microarray Of Dendritic Cells Infected By Enterovirus 71 And Endocytosis Analysis

ObjectivesTo investigate the mouse dendritic cells(DCs)maturation and the changes of gene expression profile after treated with enterovirus 71(EV71)in vitro,and to further identify possible mechanisms of DCs after EV71 infection.MethodsEV71 virus were amplified by RD cells,then we establish a model of DCs infected with EV71 virus.The immunophenotypic changes of DCs were detected by flow cytometry.Agilent mouse gene expression microarray were used to screen differential genes and the results were verified by RT-PCR.The differentially expressed genes were analyzed by GO and KEGG enrichment,and bioinformatics analysis was performed to determine the biological functions or pathways of differential genes.The expression level of VP1,which was surface protein of EV71,was detected by Western blot,and the endocytic pathway inhibitor QS11 was added to analyze the role of endocytic pathway in EV71 infection.ResultsAfter 24 hours of infection with EV71 virus,the DCs cells showing typical cytopathic effects.The results of flow cytometry showed that the positive rate of CD80 and CD86,which are characteristic markers of DCs after infected by EV71,was significantly increased,indicating that EV71 could significantly induce the maturation of DCs.Microarray gene analysis showed that 87 differentially expressed genes were screened in EV71-infected DCs,including 66 up-regulated genes and 21down-regulated genes.Six genes(Arap1,cb1 c,jun,Thbs1,cdc37,and ubtd1)were selected to verify the chip results.The RT-q PCR data were consistent with the chip results.GO analysis showed that the dysregulated genes involved 253 biologicalprocesses,of which 175 biological processes were significant.KEGG analysis showed that endocytosis pathway,TGF-? signaling pathway and T cell receptor signaling pathway were related to differentially expressed genes.Western blot results showed that after the EV71 infected DCs cells were added with the inhibitor QS11,the expression of VP1 in the DCs of the blank control group was significantly lower than that of the EV71 infected group.ConclusionWe established a model of EV71-infected mouse immature DCs.The typical CPE of DCs cells after EV71 infection indicated that DCs are sensitive cells for EV71 infection,and EV71 can induce DCs to mature.Bioinformatics analysis of the expression profile showed that the cell changes caused by EV71 infection of DCs involved 253 biological processes,of which 175 biological processes were of significant significance,mainly involving multiple processes such as viral transcription,apoptosis,and immune cell differentiation.KEGG analysis showed that endocytosis pathway,TGF-? signaling pathway and T cell receptor signaling pathway were related to differentially expressed genes.These significant molecular biological processes and their constituent genes may be related to the molecular mechanism of EV71-infected DCs cells.The enrichment of differential genes was most significant in the endocytosis pathway.Further confirmation by Western blot showed that DCs may transport EV71 virus through the endocytosis pathway.