Role And Mechanism Of Respiratory Syncytial Virus NS1 Protein In Glucocorticoid Insensitivity

PART I RSV INFECTION REDUCE GR MRNA AND PROTEN EXPRESSION LEVELObjectives RSV is the most common viral pathogen causing lower respiratory tract infection in infants under 5 years old worldwide.There is still no effective vaccine and drug prevention.Inflammation induced by RSV infection is insensitive to glucocorticoid,and the mechanism is not ful ly understood.Glucocorticoids exert its anti-inflammatory effect by regulating the glucocorticoid receptor(glucocorticoid receptors,GR)expression or transporting.The insensitivity mechanism of glucocorticoid related to GR is usually containing: decreased GR expression;inhibited GR nuclear translocation;reduced nuclear DNA binding.It is not clear whether RSV infection downregulates the expression of GR,leading to insensitivity of glucocorticoid.Therefore,the aim of this study is focus on the effect of RSV infection on GR expression level.Methods NPAs of hospitalized children with RSV infection on the first day and NPAs of the normal control group were collected to detect the mRNA and protein expression of GR.RSV or UV-RSV infected with A549 cell or BEAS-2B cell,at indicated time,collect samples to detect the mRNA and protein expression of GR.Results 1)GR mRNA and protein expression were decreased in NPAs from RSV infected infants;2)GR mRNA and protein expression were decreased in time-dependent manner both in RSV infected A549 or BEAS-2B cells.3)There is no effect of UV-RSV on GR mRNA or protein expression.Conclusions RSV infection may reduce GR mRNA and protein expression level.PART ? RSV INFECTION DECREASED GR MRNA EXPRESSION VIA UPREGULATING MIR-29AObjectives There are many miRNA binding sites in 3 'UTR of GR,so its expression can be regulated by miRNA.miRNA is a 22nt-sized non-coding RNA,which can regulate the 3'UTR of genes and mediate the stability of its mRNA.We first found that the position 1078-1084 of 3 'UTR of GR was the target sequence of miR-29 a through software prediction.mir-29 a is an epithelial cell derived miRNA,and it has been reported that RSV infection can increase the expression of miR-29 a and decrease the level of GR mRNA,so we hypothesis that RSV reduced GR mRNA level by up-regulating the expression of miR-29 a.Methods Detect the miR-29 a expression in NPAs and analysis the correlation between miR-29 a and GR mRNA or GR protein.Detect the miR-29 a expression both in RSV infected A549 cells and BEAS-2B cells.Knockdown or overexpressed miR-29 a,detect the GR mRNA or GR protein after RSV infection.Further detect whether GR gene is the target of miR-29 a with dual-luciferase reporter system.Results 1)RSV infection increased miR-29 a expression,and miR-29 a showed a significance correlation with GR mRNA and GR protein;2)live RSV increased miR-29 a expression in a dose-and time-dependent manner;3)inhibited miR-29 a partly rescued the decreased GR mRNA and GR protein by RSV.4)GR is a target gene of miR-29 a.Conclusions RSV infection reduced GR mRNA expression via up-regulating miR-29 a.PART ? RSV INFECTION DECREASED GR PROTEIN EXPRESSION VIA MEDIATING P53-MDM2 AXLEObjectives Inhibition of miR-29 a expression only can partially rescue the reduced GR protein by RSV infection,which suggested that there is another pathway for RSV to reduce GR protein.Under the cells are in hypoxia,the degradation of GR protein mainly via Mdm2-mediated ubiquitination degradation by interacting with p53 and Mdm2.RSV infection induced host cells in hypoxia,so we hypothesis that RSV reduced GR protein expression by regulating the p53-Mdm2 axis.Methods Detect the p53 protein expression in NPAs and analysis the correlation between p53 and GR protein.Detect the p53 and Mdm2 protein expression and the protein interaction among GR,p53 and Mdm2 both in RSV infected A549 cells and BEAS-2B cells.Inhibited Mdm2 expression,Mdm2 phosphorylation level and the interaction between p53 and Mdm2,detect the GR mRNA or GR protein expression and the protein interaction among GR,p53 and Mdm2.Results 1)p53 protein expression in NPAs was decreased in RSV infected infants,and was significantly correlated with GR protein expression.2)After A549 cells were infected by live virus RSV,p53 decreased in a time-dependent manner.3)Inhibition of Mdm2 expression level,phosphorylation level and interaction with p53 can partially rescue reduced GR protein by RSV.Conclusions RSV infection reduced GR protein expression via mediating p53-Mdm2.PART IV RSV NS1 PROTEIN DECREASED GR MRNA AND PROTEIN EXPRESSION VIA UP-REGULATING MIR-29 A AND MEDIATING P53-MDM2 AXLEObjectives NS1 gene,located at the 3 'end of RSV genome,is the most expressed gene and protein in the early stage and mediates a variety of biological functions.RSV NS1 protein can up-regulate miR-29 a to decrease of IFNR1,and influenza NS1 protein can mediate the degradation of p53.Whether NS1 protein is the most important protein to reduce GR expression in RSV infection,and whether NS1 mediates GR reduction by regulating miR-29 a and p53-Mdm2 is not clear.Therefore,the aim of this study was to confirm whether NS1 could reduce GR expression,and further confirm whether it could decrease GR expression by regulating miR-29 a and mediating the p53-Mdm2 axis.Methods A549 cell model of RSV infection was established to determine the expression levels of 10 genes encoded by RSV at indicated post infection time.Knockdown NS1 gene by si RNA after RSV infection of A549 cells,detect the GR mRNA,GR protein,miR-29 a and p53 protein expression level.Knockdown of NS1 gene: overexpressed miR-29 a,detected the GR mRNA,GR protein and FKBP51 and GILZ mRNA expression;overexpressed Mdm2,detected the p53 protein,GR protein,FKBP51 and GILZ mRNA expression level.Further detected the NS1 gene expression in NPAs and analysis its correlation with GR mRNA,GR protein,miR-29 a and p53 protein expression.Results 1)NS1 gene was the most expressed among the 10 genes encoded by RSV at early RSV infection of A549 cells;2)Knockdown of NS1 gene increased the GR mRNA,GR protein,p53 protein expression and decreased miR-29 a expression;3.knockdown of NS1 gene: overexpression of miR-29 a can reduce the expression of GR mRNA,GR protein,FKBP51 and GILZ mRNA;overexpression of Mdm2,decreased p53 protein,GR protein,FKBP51 and GILZ mRNA expression level.Conclusions RSV NS1 reduced GR protein expression via up-regulating miR-29 a and mediating p53-Mdm2 axle.