Construction And Identification Of A CDNA Library Of Host MRNA Targets For MiR-M4-5p Encoded By Marek's Disease Virus

Marek's disease virus(MDV)is one of the oncogenic herpes viruses that can induce tumors in their hosts,which is considered to be an excellent model for investigating the biology,genetics,and immunology of tumorigenesis.Marek's disease(MD)has affected poultry health worldwide and is responsible for huge economic losses.In the past few decades,MDV-1's virulence has increased due to imperfect immunization by vaccines.Highly virulent MDV1 strains is a great threat.So it is important to study the molecular mechanisms that trigger the interactions between virus and host in MDV-1 pathogenesis and oncogenesis.Micro RNAs(mi RNAs)are single-stranded RNA(22-24 nt)and they can regulate gene expression through post transcriptional mechanisms.Mi RNAs play different important roles in biological processes,including cellular differentiation,apoptosis,development and proliferation.Recently,a lot of micro RNAs(mi RNAs)have been identified in MDV genomes and mi R-M4-5p encoded by MDV-1 has been identified as a viral analog of cellular mi R-155.Since mi R-155 is associated with several cancers,so mi R-M4-5p was suggested to play a critical role in MDV oncogenesis.The present work was performed to construct a c DNA library and to primarily screen the putative targets for mi R-M4-5p.All hybrid PCR products amplified from CEF RNA were harvested and inserted to p MD19-T vectors,transformed into E.coli JM109 to produce a pool.The clones were selected and sequenced.The target sequences were analyzed by the online basic local alignment search tool(BLAST).A total of 88 candidate genes were obtained.Among them,29 genes contained mi RNA binding sites in 3'-UTRs and were complementary to the seed sequence of mi R-M4-5p.After three rounds of dual fluorescence reporter assays(DLRA),five host genes including PRICKLE1,COLA,BCAT1,ANTXR1 and TECPR1 were primarily filtered out.The expression level of target genes in CEFs over-expressing mi R-M4-5p or GX0101-infected was analyzed by q TR-PCR.The dataconfirmed that BCAT1 and ANTXR1 can be recognized and down-regulated by mi R-M4-5p.Our work provides an important basis for further studies on the molecular regulatory mechanism mediated by mi R-M4-5p.