Screening Of Differentially Expressed Mirnas In DEF Infected With DTMUV And MiR-221-3p Facilitate DTMUV Replication By Targeting SOCS5 Gene

Duck Tembusu virus?DTMUV?,a member of the Flaviviridae family,could infect many kinds of waterfowl and cause egg drop.The disease spreads rapidly and has caused siginificant economic loss to the waterfowl industry.microRNAs?miRNAs?are a class of small non-coding RNAs about 22nt in length that regulate gene expression at post-transcriptional level.miRNAs are involved in many biological processes such as immune response and viral infection.However,the biological functions of miRNAs in DTMUV-infected duck embryo fibroblast?DEF?has not been reported so far.Therefore,in this study,the miRNAs expression profiles in DEF cells with DTMUV infection were obtained by high throughput sequencing.Significant differentially expressed miR-221-3p was selected as a subject of study,and the effects of miR-221-3p and its target on DTMUV replication were investigated.1 Screening of differentially expressed miRNAs in DEF infected with DTMUVDEF was infected with DTMUV CQW strain,after 36h,the cells were collected and RNA was extracted.287 known miRNAs and 63 novel miRNAs were obtained by Illumina high-throughput sequencing.The expression of 26 known miRNAs and 22 novel miRNAs were changed in the infected groups.Among these miRNAs,37 miRNAs were up-regulated and 11miRNAs were down-regulated.GO enrichment and KEGG pathways analysis showed that the predicted target genes of 48 differentially expressed miRNAs were involved in several signaling pathways such as TGF-?signaling pathway,mTOR signaling pathway and p53 signaling pathway.9 miRNAs were randomly selected for qRT-PCR analysis.The result was consistent with the sequencing result.2 Study on the miR-221-3p enhance DTMUV replication via targeting SOCS5?1?Verification of the targeting relationship between miR-221-3p and SOCS5 genePrediction result showed that SOCS5 was a potential target of miR-221-3p.The target relationship was verified by dual-luciferase reporter analysis.The results showed that the relative luciferase activity was significantly reduced in mimic and pmirGLO-SOCS5-Wt transfected cells?P<0.001?,the relative luciferase activity was significantly increased in inhibitor and pmirGLO-SOCS5-Wt transfected cells?P<0.01?.Whereas mimic and inhibitor failed to significantly change the relative luciferase activity in pmirGLO-SOCS5-Mut transfected cells?P>0.05?.This results indicated that miR-221-3p could specifically bind to the3'-UTR of SOCS5.The effect of miR-221-3p on SOCS5 expression was further detected by qRT-PCR and western blot.Results showed that overexpression of miR-221-3p could decrease SOCS5 mRNA and protein expression?P<0.01?,whereas inhibition of miR-221-3p could improved SOCS5 mRNA and protein expression?P<0.01?.These results indicated that miR-221-3p could inhibit SOCS5 expression at mRNA and protein levels via targeting SOCS.?2?DTMUV replication was inhibited by target gene SOCS5ShRNAs against SOCS5 were synthesized and pcDNA3.1?+?-SOCS5 plasmid was constructed,then transfected into DEF and infected with DTMUV.We detected the effect of SOCS5 on DTMUV replication by qRT-PCR,western blot and TCID₅₀.The results showed that the viral RNA copies,viral E protein expression and viral titer were significantly increased after inhibition of SOCS5 by transfecting SOCS5-shRNA?P<0.05?.However,these three indicators were significantly reduced after overexpression of SOCS5 by transfecting pcDNA3.1?+?-SOCS5?P<0.05?.These results indicated overexpression of SOCS5 could inhibit DTMUV replication and knockdown of SOCS5 could enhance DTMUV replication.?3?miR-221-3p enhance DTMUV replication via targeting SOCS5 genemiR-221-3p mimic and inhibitor were transfected into DEF,then infected with DTMUV.The effect of miR-221-3p on DTMUV replication were detected by qRT-PCR,western blot and TCID₅₀.Results showed the viral RNA copies,viral E protein expression and viral titer were significantly increased after overexpression of miR-221-3p via transfecting mimic?P<0.05?.But these three indicators were significantly decreased after inhibition of miR-221-3p via transfecting inhibitor?P<0.05?.Results proved that overexpression of miR-221-3p could enhance DTMUV replication and inhibition of miR-221-3p could inhibit DTMUV replication.In summary,this study found the expression of 48 miRNAs were changed in DTMUV-infected DEF.Among these miRNAs,significant differentially expressed miR-221-3p could facilitate DTMUV replication by inhibit SOCS5 expression.