Screening And Identification Of Host MRNA Targets For The Viral MicroRNA MiR-M11-5p Encoded By Marek’s Disease Virus

Marek’s disease virus(MDV)is an important member of the herpesvirus.It causes Marek’s disease(MD),a typical immunosuppressive and rapid-onset T-cell lymphomagenesis disease in chickens.MicroRNA(miRNA)is a class of non-coding small RNAs in length of 19~24 nt,and can inhibit the expression of target mRNAs by the pairing of 2~7 nucleotides at the 5’ end of the seed sequence with the 3’-UTRs of the target genes.It has been previously found that the MDV genomes encode a large number of viral miRNAs.MDV-1 encodes 14 pre-miRNAs producing 26 mature miRNAs,which may play important roles in virus replication,latent infection and/or tumorigenesis.Previous in vivo dynamic expression profiling of these viral miRNAs revealed that miR-M11-5p is closely related to the pathogenesis and tumorigenesis processes of MDV-1.The pathogenicity analysis of the viruses with deletions of each miRNA of the Mid-cluster showed that the tumorigenicity of the miR-M11-5p deleted virus strain was significantly enhanced compared to the parental strain GX0101,indicating that miR-M11-5p might play a potential role as a tumor suppressor in the process of MD tumorigenesis.Therefore,the molecular mechanism mediated by miR-M11-5p in MD tumorigenesis deserves further study.To further reveal the regulatory mechanism mediated by miR-M11-5p in MD oncogenesis,we have presently performed a hybrid-PCR to amplify the candidate host target genes using the cDNA derived from chicken embryo fibroblasts(CEF)cellular RNA as template.The PCR products were ligated into the pMD19-T vector and transformed into E.coli JM109 to construct a cDNA library for screening the candidate targets for miR-M11-5p.Based on PCR identification,DNA sequencing and BLAST analysis,a total of 77 candidate genes were obtained,of which 37 binding sites recognized by miR-M11-5p were located in the 3’-UTRs of the mRNA genes.A psiCHECK-2 recombinant vector containing candidate target gene binding sites of the 3’-UTR was constructed,and co-transfected with miR-M11-5p mimics into HEK 293 T cells for analysis by dual-luciferase reporter assay.The results showed that the psiCHECK-2-3’-UTR plasmids of the candidate target genes MAFB,LOC776816 and RFX7 were significantly inhibited by miR-m11-5p mimics.Then,we constructed psiCHECK-2-mut-MAFB-3’-UTR,psiCHECK-2-mut-LOC776816-3’-UTR and psiCHECK-2-mut-RFX7-3’-UTR,together with the laboratory-preserved mut-miR-M11-5p mimics,were co-transfected with HEK 293 T cell.The results showed that the mutated miR-M11-5p lost the activity of inhibiting the three 3’-UTR reporter genes MAFB,LOC776816 and RFX7,and the 3’-UTR mutation was not inhibited by miRM11-5p.mRNA expression levels of MAFB,LOC776816 and RFX7 were detected by RT-qPCR.The results showed that miR-M11-5p significantly down-regulates the expression levels of mRNA of these three candidate target genes.These results indicate that the three host genes,MAFB,LOC776816 and RFX7,are host target genes for miR-M11-5p.Our data provides an important foundation for further elucidation of the regulatory mechanism mediated by miR-M11-5p in MDV tumourigenesis.