Study On The Effect And Mechanism Of MiR-142a-3p Regulating PHEV Replication

Porcine hemagglutinating encephalomyelitis virus(PHEV)belongs to the genusβ Coronavirus of the family Coronavirus and causes clinical symptoms such as neurological symptoms,vomiting,and diarrhea in piglets under 3 weeks of age.PHEV has typical characteristics of neurotropic coronavirus,but the mechanism of nerve damage caused by this virus is still unclear.It is well known that mi RNAs involves in the pathogenesis of various viruses by specifically regulating their target genes.In our previous research,the brain tissue of PHEV-infected mice was detected by gene chip technology,and the expression levels of mi R-142a-3p and other mi RNAs were significantly changed.In this study,mi R-142a-3p with significant differential expression was selected,and its effect on PHEV replication was well studied,which provided a theoretical basis for the in-depth investigation of the PHEV pathogenesis.In this study,the expression of mi R-142a-3p in PHEV-infected models was detected by real-time PCR(q RT-PCR)in vitro and vivo.The results showed that mi R-142a-3p was significantly up-regulated after viral infection compared with the control group.Consistented with the gene chip results,suggesting that it may be involved in the process of PHEV infection.Subsequently,N2 a cells were transfected with chemically synthesized mi R-142a-3p mimic,following by virus inoculation.QRT-PCR and western blotting(WB)were used to detect the level of PHEV m RNA and protein in cells.The mi R-142a-3p mimic transfecting cells showed significant up-regulation in the expression of PHEV m RNA and protein,comparing with the control group.The above experiments confirmed that mi R-142a-3p is involved in the PHEV infection and promotes viral replication.To further clarify the mechanism of mi R-142a-3p involved in PHEV replication,we used Target Scan,mi RBase,Pic Tar,and other bioinformatics websites to predict and screen mi R-142a-3p target genes.Finally,protein Rab3 a,a vesicle transport related protein was predicted as potential target gene.To detect whether Rab3 a is a mi R-142a-3p specific target gene,the Rab3a-MUT(mutant)and Rab3a-WT(wildtype)dual fluorescence reporter vector were constructed.The results of the dual luciferase reporter system showed that mi R-142a-3p could bind to the Rab3 a 3’UTR region and verified that Rab3 a is a target gene of mi R-142a-3p.Next,different doses of mi R-142a-3p mimic or inhibitor were transfected into N2 a cells.It was found by q RT-PCR and WB that mi R-142a-3p negatively regulated Rab3 a expression level in a dose-dependent manner.In addition,combined with Indirect Immunofluorescence(IFA)detection result further confirmed that Rab3 a is a target gene of mi R-142a-3p.However,whether mi R-142a-3p participates in PHEV replication through Rab3 a needs further verification.In order to verify that Rab3 a is an important protein of mi R-142a-3p regulating PHEV replication,this experiment constructed the Rab3 a overexpression vector(Rab3a-GFP)and Rab3 a si RNA,followed by inoculation of virus.The expression of virus was detected by q RT-PCR and WB.The results showed that the expression of PHEV m RNA and N protein were significantly up-regulated after transfection of Rab3 a si RNA in N2 a cells.And the viral expression was significantly inhibited after transfection of Rab3 a overexpression vector.All the results of this experiment not only indicate that Rab3 a plays a negative regulatory role in PHEV replication,but also further confirm that mi R-142a-3p can affect PHEV replication by regulating Rab3 a expression.In summary,the level of mi R-142a-3p was significantly up-regulated during PHEV infection,which inhibiting the expression of Rab3 a,thereby promoting the replication of PHEV in the mouse cells.All the results of this study not only provide experimental support for mi RNAs involved in the regulation of PHEV replication,but also provide the basis for in-depth discussion of PHEV pathogenesis and treatment options.It has important scientific significance.