The Mechanism Of MiR-140-5p Regulating HDAC4 To Participate In CV-A10 Immune Escape

Enterovirus A71(EV-A71)and Coxsackievirus A16(CV-A16)have been identified as important pathogens of Hand-foot-mouth disease(HFMD),but in recent years,the cases of hand,foot and mouth disease caused by Coxsackievirus A10(CV-A 10)and Coxsackie virus A6(CV-A6)are on the rise.Studies have shown that CV-A 10 and CV-A6 have gradually become the dominant pathogens of hand-foot-mouth disease.The pathogenic mechanism of hand-foot-mouth disease caused by CV-A 10 infection is not clear,so there is an urgent need to carry out research on the pathogenesis of CV-A10-related hand-footmouth disease.Based on the results of previous studies,microRNA expression profile analysis in human bronchial epithelial cells(human bronchial epithelial cells,16HBE)infected with EV-A71 and CV-A 16 showed that the expression of miRNA-140-5p was up-regulated in EV-A71 and CV-A 16 infection.The bioinformatics software Targetscan was used to predict that histone deacetylase 4(HDAC4)may be one of the genes targeted by miRNA140-5p.HDAC4 is an antiviral regulatory factor,which plays an important role in the regulation of virus infection and host immune response.Interferon is produced when the virus infects the host,in which type Ⅰ interferon can induce JAK-STAT(Janus KinaseSignal Transducer and Activator of Transcription)signal pathway and the expression of downstream interferon stimulating factor to mediate antiviral immune response,but at the same time,the virus will evolve some escape mechanisms to interfere with or destroy the interference signal pathway so as to sustain infection.It has been confirmed that HDAC4 can not only affect the antiviral response of interferon by regulating the activity of interferon regulatory factor 3(IRF3),which mediates interferon production,but also affect the JAK-STAT signal pathway and the expression of downstream interferon stimulating genes.Therefore,we speculate whether HDAC4 participates in virus infection and immune escape by regulating the corresponding mechanism in CV-A 10 infection.First of all,the proliferation kinetics of CV-A 10 in 16HBE and Vero cells was analyzed by micropore cytopathic method.It was found that CV-A 10 could replicate and infect 16HBE cells in the same way as Vero cells with inherent IFN α/β gene deficiency.It is speculated that this may be due to the fact that CV-A 10 escapes the antiviral effect of interferon by some mechanisms.Secondly,the expression of important regulatory factors in interferon signal pathway was detected by cellular immunofluorescence experiment and Western Blot.The results showed that after CV-A 10 infection,the expression of IRF3 and STAT1 decreased,and the expression of IFI44 increased at first and then decreased significantly.Then,the expression of miRNA-140-5p was up-regulated and the expression of HDAC4 was down-regulated in CV-A10-infected cells by qRT-PCR,and the dual luciferase reporter gene detection system was used to verify that miRNA-140-5p could target and regulate HDAC4.Further immunofluorescence and Western Blot detection showed that the expression of HDAC4 protein decreased after CV-A10 infection.Finally,in order to further explore the effect of HDAC4 on the expression of important regulatory factors in interferon signal pathway,the virus was reinfected by transfection of overexpressed HDAC4 plasmid and empty plasmid.Compared with the control group,it was found that the expression of HDAC4 did not decrease significantly after overexpression of HDAC4,while the expression of IRF3,phosphorylated IRF3 in total protein and nuclear IRF3 maintained at a high level.The expression of STAT1,phosphorylated STAT1 and STAT2 in the total protein increased,and the expression of IFI44 remained at a high level.At the same time,it was also detected that the VP 1 expression of CV-A10 decreased significantly after overexpression of HDAC4 and reinfection of the virus.To sum up,this study concluded that after CV-A10 infection,the expression of miRNA-140-5p was up-regulated,which decreased the expression of HDAC4 through targeted regulation of HDAC4,while the down-regulation of HDAC4 reduced the nuclear localization of IRF3,the phosphorylation level of IRF3/IRF7,and the STAT1 and STAT2 proteins in JAK-STAT signaling pathway,which inhibited the expression of interferon and made CV-A10 escape the host’s innate immune response.