Prediction, Screening And Identification Of The Host MRNA Targets For MiR-M12-5p/3p Encoded By Marek's Disease Virus

Marek's disease(MD)is an immunosuppressive disease and a tumorous disease characterized by solid organ tumors caused by Marek's disease virus(MDV).MD is currently known as the first virus disease that can be prevented by vaccine immunization,and it has a very typical kinetic process of tumor development.Therefore,MDV provides a natural virus-host interaction model for the development of virus-encoded mi RNAs.Previous studies have found that the infection of the strain of mi RNA precursor single gene deletion of Meq-cluster encoded by MDV-1,chicken mortality and tumor incidence was significantly reduced.The results indicate that mi RNAs of Meq-cluster may have the function of regulating the pathogenic phenotype of MDV.Compared with mi R-M12,mi R-M4,mi R-M9 and mi R-M5 precursor single-gene deletion,mi R-M12 precursor single gene deletion of the strongest role,suggesting that mi R-M12 coding of mature mi RNAs may play an important regulatory role in the development of MD tumors.In order to study the mechanism of mature mi R-m12-5p and mi R-m12-3p encoded by mi R-M12 in MD tumorigenesis,RNAhybrid which is the bioinformatics software was used to predict the host target gene of mi R-M12-5p and mi R-M12-3p.A total of 19 binding sites between mi RNA and m RNA were obtained from 14 candidate target genes which came form 45 down-reguated genes.Nine target genes was selected and tested.The HEK 293 T cells were co-transfected with the psi CHECKTM-2 vector which recombinant of the candidate target gene and the pc DNATM6.2 vector constructed with mi R-M12.And then use the dual luciferase reporter assay to verify the in vitro interactions of mi R-M12-5p/3p and target genes.The results showed that mi R-M12-5p could down-regulate the expression of HVCN1 and mi R-M12-3p could down-regulate the expression of HSPA4.The seed sequences of mi R-M12-5p and mi R-M12-3p and the binding sites of HVCN1 and HSPA4 were mutated to verify whether the interaction of mi RNA: m RNA was site-specific.The results showed that mut-mi R-M12-5p could not inhibit the renilla luciferase activity of psi CHECK-2-HVCN1-3'-UTR after mutations in the mi RNA seed region.Mut-mi R-M12-3p could not inhibit the renilla function of psi CHECK-2-HSPA4-3'-UTR.The mi R-M12-5p could not inhibit the activity of renilla luciferase in psi CHECK-2-mut-HVCN1-3'-UTR and the mi R-M12-3p could not be inhibited after the mutation of HSPA4 binding site after mutation of HSPA4 binding site.The results showed that mi R-M12-5p and mi R-M12-3p inhibited the expression of HVCN1 and HSPA4 mainly through specific binding sites.Finally,CEFs were infected by mi R-M12 gene deletion,and the expression of m RNA of candidate target gene was detected in vivo.Real-time fluorescence quantitative PCR results showed that mi R-M12-5p could downregulate the expression of HVCN1 and mi R-M12-3p could down-regulate the expression of HSPA4.The results of the above studies show that mi R-M12-5p can target regulation the expression of HVCN1;mi R-M12-3p can target the expression of HSPA4.MiR-M12-5p can downregulate the expression of HVCN1 and mi R-M12-3p can down-regulate the expression of HSPA4 by bioinformatics prediction and experimental verification.This study laid a foundation for further revealing the molecular mechanism of mi R-M12-encoding mi RNAs that control MDV pathogenesis and tumorigenesis.