Identification Of Targeted Gene And Preliminary Function Of PRV-encoded Prv-miR-LLT11a

Micro RNAs(miRNAs)are the small endogenous regulatory noncoding RNAs of approximately 22 nucleotides in length that play an important role in the post-transcriptional regulation of gene expression?miRNAs are the crucial factor in a diverse biological processes such as antiviral defence,oncogenesis,apoptosis,metabolism,differentiation and development in higher eukaryotes.Mature miRNA mediates the gene expression either by inducing targeted mRNA decay or translational repression after binding to complementary target sequences located in the 3'untranslated region(3'UTR),.Researches show that miRNAs play a critical role in host-virus interaction.The long latency-associated transcript(LLT)encodes 11 mature miRNAs including prv-miR-LLT11 a in porcine kidney(PK-15)cells infected by the pseudorabies virus(PRV),nevertheless the temporal expression and funtion of these miRNAs are unknown.The paper aims to detect the temporal expression,identify the targeted gene and revel the function in immune regulation of PRV-encoded prv-miRLLT11 a.1.The temporal expression profile of prv-miR-LLT11 a in PK-15 cells infected by the pseudorabies virusThe temporal expression profile of prv-miR-LLT11 a in PK-15 cells infected by the pseudorabies virus was detected by stem-loop RT-qPCR to provide theoretical basis for the further study of target gene.PK-15 cells infected with PRV QBA wild strain isolated from our laboratory were were harvested at 0,1,2,4,6,8,12,24 hours post infection(hpi)and used for small RNAs extraction.Stem-loop RT-PCR was adopted to verify the temporal expression of prv-miR-LLT11 a.the results show that the expression of prv-miR-LLT11 a increased remarkably significantly in PK-15 cells at 1 hour infection with PRV QBA strain,then decreased significantly to the lowest level at 6 hpi,and the expression level increased significantly eight hours later,which showed a extremely significant differential expression.The results indicate that prv-miR-LLT11 a may play an importan role in gene regulatory networks during PRV infection.2.prv-miR-LLT11 a could target and inhibit the expression of SLA-1 geneIn order to study the regulatory function of prv-miR-LLT11 a,targeted gene of prvmiR-LLT11 a was predicted using the online targeted gene prediction tool RNAhybrid,the results showed that it could target Major histocompatibility complex class gene?(also known as Swine leukocyte antigen 1 gene,SLA-1)of Sus scrofa in vitro.To study the interaction of prv-miR-LLT11 a and SLA-1 in vitro,the 3'UTR and 3'UTR mutation of SLA-1 was amplified and cloned into dual luciferase reporter vector pmir GLO,constructed eukaryotic expression vector of SLA-1 3'UTR and SLA-1 3'UTR mut.Dual luciferase reporter assay system was used to detect the interaction between prv-miRLLT11 a and SLA-1 gene.The results showed that prv-miR-LLT11 a could target the SLA-1 gene of Sus scrofa.The prv-miR-LLT11 a mimics was transfected into PK-15 cells and the effect of prv-miR-LLT11 a overexpression on the expression of SLA-1 at transcriptional and translational level was detected by RT-qPCR and Western Blot,respectively.The results of RT-qPCR showed that prv-miR-LLT11 a could significantly decrease the transcription of SLA-1 gene,and also had a certain inhibitory effect on protein level.In order to verify the regulation of prv-miR-L-LT11 a targeted SLA-1 gene further,prv-miR-LLT11 a mimics were transfected with different doses and the results show that the inhibitory effect of prv-miR-LLT11 a targeted SLA-1 gene was not dosedependent on the transcriptional and translational levels.The regulation expression of prv-miR-LLT11 a targeted SLA-1 gene whether affects the function of antigen presentation is needed to be studied further.3.Effects of prv-miR-LLT11 a overexpression on the expression of PLC,and the proliferation of PRVPeptide loading complex(PLC)is composed of TAP,tapasin,CRT,CNX,ERp57,?2m and each component of this complex are synergistic with MHC I molecule and transport polypeptide to the cell surface for the CD8+T cells identification,and play an important role in the process of cellular immune function in the body.In view of the regulation of prv-miR-LLT targeted SLA-1gene and the synergistic effects of SLA-1 molecule and PLC in the process of antigen transport,the aim of this study was to investigate the effects of prv-miR-LLT11 a overexpression on the expression of PLC and the multiplication of PRV.RT-qPCR and viral plaque assay were used to detect the effect of prv-miR-LLT11 a overexpression on the expression of PLC and the multiplication of PRV.The results show that prv-miR-LLT11 a overexpression could significantly inhibit the expression of TAP1 gene and multiplication of PRV,but had no obvious effect on the expression of the other PLC gene.It is suggested that PRV-encoded miRNAs may affect the host immune response by regulating the antigen transport pathway from cytoplasm to endoplasmic reticulum and the expression of the related gene.