Identification Of MiR-140 Target Gene And Its Role In CSFV Replication In Swine Umbilical Vein Endothelial Cell

Classical swine fever virus(CSFV)is a single-stranded positive-stranded RNA virus.The genome length of the coding region of CSFV is about 12.3 kb,which is translated into 12 polyprotein.MicroRNAs(miRNAs)are non-coding single-stranded RNAs that exist in many biological cells.MiRNAs are about 22 nucleotides(nt)in length and mainly perform RNA splicing and pre-transcriptional regulation.MiRNAs play a role through targeting mainly the 3'untranslated regions(3'UTR)of target genes.When the seed sequence of miRNAs is complementary completely to the target gene sequence,miRNAs produce a marked effect through target degradation,and when they are not completely complementary,miRNAs work through translation inhibition.Our differential miRNAs screening of CSFV infected swine umbilical vein endothelial cells(SUVEC)by genomic high throughput sequencing revealed that there were 8 up-regulated and 4 down-regulated miRNAs,but the role of these miRNAs in CSFV was not yet studied.Software prediction shows that there are target genes regulated by miR-140 in both CSFV and SUVEC.Identification of target genes regulated by miR-140 in SUVEC,identification of interaction proteins targeted by miR-140 and CSFV and their effects on CSFV replication will provide new ideas and new scientific data for the study of pathogenesis of CSFV.The main results of this study are as follows:(1)CSFV down-regulated miR-140 in swine umbilical vein endothelial cellsMiR-140 was detected by RT-qPCR at 72 h after CSFV infected SUVEC.The results showed that miR-140 was significantly down-regulated in SUVEC(p < 0.001).(2)miR-140 inhibited the replication of CSFV in swine umbilical vein endothelial cellsThe mimics and inhibitors of miR-140 were transfected into SUVEC,and CSFV was inoculated at 12 h after transfection.The transcription of viral gene in cell and the copy of viral gene in cell supernatant were detected by RT-qPCR,respectively at 24 h,36 h,48 h and 72 h after infection.The results showed that the amount of CSFV at the levels of nucleic acid decreased significantly(p < 0.05)in the cells transfected with miR-140 mimics and showed dose-dependent.The cells transfected with the miR-140 inhibitors showed that the viral nucleic acid content in the cells and viral copies in the supernatants of the experimental group was significantly higher than that of the control group(p < 0.05).(3)The target gene of miR-140 was Rab25 in swine umbilical vein endothelial cellsThe regulation of Rab25 genes by miR-140 was detected by double luciferase reporting system.The results showed that the activity of luciferase/renin luciferase in cells transfected with wild-type recombinant plasmid of Rab25-3'UTR and miR-140 mimics was significantly lower than that of wild-type recombinant plasmid of Rab25-3'UTR and miR-140 mimics NC(p < 0.001).However,there was no significant difference between Rab25-3'UTR mutant recombinant plasmid and miR-140 mimics and Rab25-3'UTR mutant recombinant plasmid and miR-140 mimics NC group.Suggesting that Rab25-3'UTR was the target gene of miR-140.The mimics and inhibitors of miR-140 were transfected into SUVEC,and the expression of Rab25 at the levels of nucleic acid and protein was detected by RT-qPCR and Western blot at 24 h after transfection.The results showed that miR-140 could significantly down-regulate the expression of Rab25(p < 0.001).The expression of Rab25 was significantly up-regulated when miR-140 inhibitors were transfected into cells(p < 0.05).(4)Rab25 promoted CSFV replicationThe recombinant plasmid of Rab25 and the RNA interference targeting Rab25 gene were constructed.CSFV was inoculated at 24 h after CMV-Rab25 and CMV empty vector were transfected into SUVEC.RT-qPCR was used to detect the nucleic acid of CSFV at 24 h and 48 h after CSFV infected.It was found that CSFV was up-regulated at nucleic acid level.There was no significant difference at 24 h,but was significant difference at 48 h(p < 0.001).CSFV was inoculated at 24 h after shRab25-2 and shN were transfected into SUVEC.RT-qPCR was used to detect the nucleic acid of CSFV at 24 h and 48 h after CSFV infected.The results showed that CSFV nucleic acid content was no significant difference between the experimental group and the control group at 24 h after exposure,but was significantly difference at 48 h.(p < 0.001).(5)Rab25 co-localized with CSFV NS5 A,and the 804-1491 bp of NS5 A may be the key siteThe pDsRed1-N1-Rab25 was co-transfected into SUVEC with pcDNA3.1(+)-NS2,pcDNA3.1(+)-NS3 and pcDNA3.1(+)-NS5 A,respectively.The co-localization of Rab25 with NS2,NS3 and NS5 A was observed by confocal scanning microscopy.The results showed that there was obvious co-localization between Rab25 and NS5 A.The pEGFP-C1-NS5A-1-84,pEGFP-C1-NS5A-84-804 and pEGFP-C1-NS5A-804-1491 were co-transfected into SUVEC with pDsRed1-N1-Rab25.The co-localization of Rab25 and three expression products was observed by confocal scanning microscopy.The results showed that there was significant co-localization between the expression products of the 804-1491 gene segment and Rab25.This study showed that the target gene of miR-140 was Rab25,which co-located with NS5 A protein of CSFV.Rab25 protein could promote the replication of CSFV.