| Seneca virus A(SVA),the member of the family Piconaviridae,is a newly emerged virus that can cause porcine idiopathic vesicular disease(PIVD).Since the clinical symptoms are similar to those of the foot-and-mouth disease,SVA has attracted the attention of pig industry all over the world.However,the pathogenesis of SVA is still unclear,especially the study on the effect of host miRNA on natural immune response after SVA infection which is still a blank.Accordingly,this study aimed to explore the role of ssc-miR-1285 in the regulation of the key molecules in the RIG-I signaling pathway and to determine the effect of the target genes in PK15 cells after SVA infection by using the fluorescence quantitative PCR and Western Blot techniques.The main results of this study were as follows,(1)Prokaryotic expression and purification of SVA VP1 protein by nickel column were performed.Then the prepared single clone antibody was proved by Western Blot and screened by IFA.Six hybridoma cell lines were obtained and No.3 and No.13 McAb were finally selected.It will provide a basis for further study of ssc-miR-1285 regulating RIG-I signaling pathway.(2)11 differentially expressed miRNAs were screened through high-throughput sequencing technologies and 7 of them(4 miRNAs up-regulated and 3 miRNAs down-regulated)were associated with RIG-I signaling pathway.The relative expression level of these seven miRNAs in PK15 cells affected by SVA were subsequently determined with RT-PCR.The results were consistent with the trends in high-throughput sequencing results that indicated the results of the high-throughput sequencing results were reliable.(3)In addition,the ssc-miR-1285,which has the most significant level of expression,was significantly up-regulated as the increase of SVA infection time.TaqMan-RT-PCR and qRT-PCR were used to analyze the effect of ssc-miR-1285 on the replication of SVA genome and the transcription level of key molecules in the RIG-I signaling pathway.Compared with the control group,ssc-miR-1285 didn't directly inhibite the replication of SVA.(4)Fluorescence quantitative RT-PCR and ELISA were used to determine the expression of IFN-? in PK15 cells after SVA infection.The results indicated that the transcription level of IFN-? was up-regulated with the increase of SVA dose and infection time,but the amount of secretion in the supernatant of cell culture was up-regulated significantly in 12 h and 18 h after infection.Further studies on IFN-? mRNA levels suggested that overexpression of miR-1285 significantly inhibited IFN-? and inhibition of miR-1285 incresed the IFN-? expression level(P < 0.05).However,opposite results were oberseved after 24 h SVA infection.(5)The results of PK15 cell cycle monitoring by flow cytometry showed that over expression of ssc-miR-1285 48 h significantly promoted cell growth and proliferation compared with the control group(P < 0.05).(6)MAVS,TRAF3,IRF3,IRF7 mRNA were significantly up-regulated(P < 0.05),while TANK and TKB1 showed a tendency of up-regulation(P > 0.05).(7)Combing bioinformatics methods were used to predict ssc-miR-1285 target sites and Western Blot and other methods were used to screen target genes of miR-1285.The results suggested that the target gene of ssc-miR-1285 was DDX3 X.Three pairs of interference RNA to DDX3 X were synthesized to further verify the function of DDX3 X,and the most effectve si-DDX3 X was selected by qRT-PCR and Western Blot.(8)The expression mRNA of key molecules in the RIG-I signaling pathway was detected after silencing the expression of DDX3 X.It was found that the expression of MAVS,TRAF3,and IFN-?was inhibited by silencing DDX3 X relative to the control group,whereas the mRNA level of TKB1,TANK were promoted.This study established the inner connection between miR-1285 and its target genes and elucidated the role of miR-1285 which could lay the foundation for exploring the molecular mechanism of miRNA regulation of SVA infection and provide new ideas and methods for SVA prevention. |
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