The Regulatory Mechanism Mediated By Mdv1-miR-M4-5p In Marek’s Disease Virus-induced Tumorigenesis

Marek’s disease virus (MDV) is one of the few oncogenic herpesviruses that caninduce tumors in their natural hosts, namely Marek’s disease (MD), which is the firsteffective use of antiviral vaccines to prevent a naturally occurring cancer. Thus, MDVinfection is considered to be an excellent model for investigating the biology, genetics,and immunology of tumorigenesis. Recently, a lot of microRNAs (miRNAs) havebeen reported in MDVs and the mdv1-miR-M4-5p was identified as a virus-encodedmiR-155analog. Since miR-155is a host miRNA associated with several cancers,mdv1-miR-M4-5p was suggested to play a critical role in MDV oncogenesis.However, the precise mechanism whereby it was involved in MD lymphomagenesisremained unknown.To investigate the potential regulation mechanism of the viral miR-155analog inMDV oncogenesis, the putative mRNA targets recognized by mdv1-miR-M4-5p werefirst predicted using the bio-softwares TargetScan and RNAhybrid. A total of200conserved candidates containing210binding sites were predicted by TargetScan fromall the5304chicken3’-UTRs while from198host genes down-regulated in MDVinfection, a total of12candidate genes were obtained utilizing RNAhybrid. Onlythree of the predicted miRNA binding sites are found in the3’-UTR of genes LatentTGF-β binding protein1(LTBP1), chemokine (C-C motif) receptor7(CCR7), andmatrix metalloproteinase13(MMP13), which are overlap between these twobioinformatic algorithms and represent as the most potential mRNA candidatestargeted by the MDV-encoded miR-155analog. For the primarily scanning of mRNAtargets, DLRA was performed to analyze the in vitro interactions between miRNA and3’-UTRs of candidate mRNAs. The relative luciferase activity of Renilla luciferasereporter with all the three3’-UTRs were significantly down-regulated bymdv1-miR-M4-5p (P<0.01), but the repression effects to parental3’-UTRs were alsoabolished when the mdv1-miR-M4-5p was mutated. The results indicated that the3’-UTRs of LTBP1, CCR7and MMP13genes can be targeted by mdv1-miR-M4-5pin vitro. After showing the down-regulation of mRNA targets in report assays, we attempted to use qRT-PCR to determine whether mdv1-miR-M4-5p regulates thetarget gene expression at the transcriptional level. The overexpression of theMDV-encoded miR-155analog in CEFs may specifically targets LTBP1rather thanCCR7or MMP13. The in vivo miRNA-mRNA target interactions were furtherinvestigated by a MDV de novo infection system using the parental virus GX0101andits mutant GX miR-M4, in which the mdv1-miR-M4-5p had been previously deletedfrom the viral genome. We found that gene silence of mdv1-miR-M4-5p up-regulatesthe expression of LTBP1at both transcriptional and translational levels.Considering the LTBPs are extracellular matrix microfibrils and mainly functionto ensure the correct folding, efficient secretion, and activation of TGF-β, wedetermined the secretion dynamics of TGF-β1in MDV-infected CEFs, in which theLTBP1expression is down-or up-regulated by mdv1-miR-M4-5p using differentMDV infections. These data indicates that during the processes of MDV infection, theviral miR-155analog regulates the secretion of TGF-β1by targeting LTBP1. Toinvestigate whether mdv1-miR-M4-5p can consequentially regulate TGF-β signalingin MDV-infected CEFs and birds, a chicken TGF-β/BMP Signaling Pathway PCRArray was employed. Among the84genes involved in the TGF-β pathway, theexpression of15genes was up-regulated, and3genes were down-regulated inGX0101-infected CEFs, compared to the GX miR-M4-infected CEFs. While thec-myc gene, a well-known oncogene, was the most significantly up-regulated gene.This was further confirmed by western blot and immunohistochemistry assay. Furthermore, we studied the functional phenotype of deregulated TGF-β signaling in CEFs.Compared to the mock transfected cell or negative control, the CEF cellviability/proliferation was significantly increased by the infection of GX0101or thetransfection of mdv1-miR-M4-5p, but the apoptosis was unaffected. Importantly, avery similar phenotype was observed in the cells transfected with LTBP1-specificsiRNAs.We have presently identified the host mRNA targets and finally identified theLTBP1as a critical target of mdv1-miR-M4-5p. We found that in MDV infection,down-regulation of LTBP1expression by mdv1-miR-M4-5p led to a significantdecrease of the secretion and activation of TGF-β1, with suppression of TGF-β signaling and a significant activation of expression of the c-Myc, a well-knownoncoprotein which is critical for virus-induced tumorigenesis. Our findings reveal anovel and important mechanism to understand how mdv1-miR-M4-5p potentiallycontributes to MDV-induced tumorigenesis.