Screening And Characterization Of The Host MRNA Targets For The MiR-M4-5p Encoded By Marek's Disease Virus

Marek's disease(MD)is an immunosuppressive disease caused by Marek's disease virus(MDV)infection.MD is the first tumor disease in the world that can be effectively controlled by immunonization with a viral vaccine,so MDV infection and tumorigenesis have been regarded as a ideal model for studying the pathogenesis of viral oncology.MicroRNA(miRNA)is a class of small non-coding RNA with the length of about 20-24 nt,which can regulate a variety of cellular biological processes,mainly by reducing the stability of mRNA,inhibiting the post-transcriptional expression level of genes and protein translation.miRNAs encoded by MDV play a crucial role in viral pathogenesis.Previous studies have found that miR-M4-5p is homologous to host proto-oncogene miR-155,and knockout of miR-M4-5p from the viral genome can significantly reduce the pathogenicity and oncogenicity of MDV-1,suggesting that miR-M4-5p may be an important regulator of MDV-induced tumorigenesis.To further reveal the regulatory mechanism mediated by miR-M4-5p in MD biology,we used CEF total cellular RNA as the template to produce a cDNA library utilizing the hybrid-PCR,for screening the host mRNA targets for miR-M4-5p.A total of 128 candidate host genes were obtained,of which there are 73 candidate binding sites were located in the 3'-UTRs and 23 of them contain the perfect matches of miRNA:mRNA.Further experiments of the dual luciferase reporter assay(DLRA)and the quantitative real-time PCR(qRT-PCR)analysis characterized three host genes,TMEM230,DPT and DCLK1,as the in vivo targets for miR-M4-5p.Our work provides an important basis for further elucidating the regulatory mechanism of miR-M4-5p in MD oncogenesis.