The Regulatory Mechanism Mediated By MiR-M2-5p In Regulating Marek Disease Virus To Promote Cell Proliferation And Inhibit Cell Apoptosis

Marek's Disease(MD)caused by Marek's disease virus(Marek's disease virus,MDV)is an infectious,tumorigenic,lymphoproliferative avian Disease.MDV is a double-stranded linear DNA virus belonging to the subfamily Alphaherpesvirinae.The continued increase in the virulence of MDV has led to MD outbreaks worldwide.This disease is the earliest virus-induced tumor disease that can be controlled by vaccine,which provides an important model for studying the biology and immunology of herpes virus-induced tumors.MicroRNAs(miRNAs)are single-stranded RNA with a length of 22-25 nucleotides,which are highly evolutionarily conserved and developmental regulatory,and are expressed in a tissue-specific manner,playing an important role in the regulation of gene expression in physiological and pathological processes.MDV1 genome encodes 26 mature miRNAs,and three distinct miRNA gene clusters are formed in the genome,which are named Meq gene cluster,Mid gene cluster and LAT gene cluster respectively.Similar to Epstein barr virus(EBV)and kaposi's sarcoma associated herpes virus(KSHV),MDV1-encoded miRNAs,particularly the Meq gene cluster,play an important role in viral replication,latency,and tumor formation.In addition to miR-M4-5p(miR-155homologous)and miR-M3-5p,we found that miR-M2-5p encoded by the Meq gene cluster may be involved in inducing T-cell lymphoma,and its potential regulatory mechanism remains to be confirmed.In order to reveal the molecular mechanism of miR-M2-5p in the regulation of MD tumor process,hybrid PCR technology combined with DNA sequencing was used in this study as a fast and efficient method to screen potential host mRNA targets of miR-M2-5p,and a library containing 51 candidate target genes was constructed.Among them,the miR-M2-5p binding site of 25 candidate target genes was located in the 3'UTR of the gene.The genes in this library are involved in signaling molecules of different network signaling pathways in cells,which provide abundant resources for the full study of the function of miR-M2-5p.Subsequently,two important host target genes,RBM24 and MYOD1,were screened and verified by experimental methods,which laid a foundation for further study on the molecular regulation mechanism of miR-M2-5p in MDV tumorigenesis.To explore the specific function of MDV1 encoded miR-M2-5p in MD tumorigenesis and elucidate its precise regulatory mechanism.We first conducted phenotypic validation of the function of miR-M2-5p in host cells,and found that miR-M2-5p could not only promote cell proliferation,but also inhibit cell apoptosis.Further mechanism studies were conducted in combination with screened miRNA target genes RBM24 and MYOD1.RBM24 is an important member of RBPs and plays an important role in post-transcriptional regulation.Our study showed that miR-M2-5p caused overexpression of p63 by mediating downregulation of RBM24 and promoted proliferation of CEF during MDV1 infection.Since p63 is generally considered to be an important oncogene,the regulation of p63 by miR-M2-5p targeting RBM24 may be one of the important ways that MDV1 triggers MD lymphoma.As another important target of miR-M2-5p,MYOD1 plays an important role in regulating cell proliferation,differentiation and even apoptosis.In this study,it was found that miR-M2-5p could directly target and inhibit the expression of MYOD1,thus inducing the down-regulation of the expressions of gga-miR-1,gga-miR-206 and gga-miR-223.On the one hand,overexpression of miR-M2-5p induced down-regulation of gga-miR-223 could inhibit the expression of IGF2 and promote the proliferation of cells.On the other hand,miR-M2-5p inhibited the expression of gga-miR-1and gga-miR-206,leading to up-regulation of Pax3,activation of two anti-apoptotic factors,Bcl-2 and Bcl-xl,and finally inhibition of cell apoptosis.The transcription of miR-M2-5p promotes the cell proliferation of and inhibits cell apoptosis by inhibiting the two target genes to regulate the different signal pathways.These data confirmed that miR-M2-5p plays a direct role in tumorigenesis.Meanwhile,it was revealed that single viral miRNA can simultaneously promote the pathogenesis and tumorigenesis of herpes virus by identifying and regulating multiple signaling pathways.