Roles Of MiRNA And MDV Oncogene Meq In The Transformation Of Chicken Marek's Disease Lymphoma

Chicken Marek's disease(MD),caused by Marek's disease virus(MDV),is a lymphotropic neoplastic disease.Previous miRNAome analysis showed that gga-miR-219b,gga-miR-130b-3p and gga-miR-140-3p were significantly downregulated in chicken MDV-induced lymphoma.In this study,we further investigated the mechanisams of these there miRNAs and MDV oncogene Meq in MD tumorigenesis.The gain/loss of function assay showed that gga-miR-219b inhibited cell migration and reduced cell proliferation by promoting apoptosis not by cell cycle arrest.Gga-miR-219b also suppressed expression of two cell invasion-related genes MMP2 and MMP9.The results indicated suppressive effect of gga-miR-219b on MD tumorigenesis.The gene B-cell chronic lymphocytic/lymphoma 11B(BCL11B)was verified as a direct target gene of gga-miR-219b through dual-luciferase reporter gene assay.RNA interference was performed to block BCL11B.As expected,the effects triggered by BCL11B downregulation were in accordance with that triggered by gga-miR-219b overexpression,suggesting that BCL11B was a stimulative regulator of MD transformation.Additionally,gene expression level of anti-apoptotic genes BCL2 and BCL2L1 was downregulated and pro-apoptotic gene TNFSF10 was upregulated in MSB1 cells with gga-miR-219b overexpression or BCL11B knockdown,which suggested gga-miR-219b promoted cell apoptosis via mediating gene expression in the apoptosis pathways.Moreover,both gga-miR-219b and BCL11B influenced the expression of Meq gene,the most important oncogene in MDV.The Meq gene is highly expressed in MD tumor tissues and MD T-lymphoblastoid cell lines.To elucidate the role of Meq in MD tumor transformation,we used RNA interference technology to block its expression,and analyzed the biological effects of Meq knockdown on MSB1 cells.A siRNA with interference efficiency of 70%was transfected into MSB 1 cells.The results showed that MSB 1 cell proliferation was inhibited remarkably post-Meq knockdown.The cell cycle was unaffected,while caspase-6 activity,which is involved in the apoptosis pathway,was upregulated significantly post-Meq knockdown.Further results showed that expression of the BCL2 gene was downregulated significantly at the translational level.MSB1 cell migration did not exhibit any obvious change,but the expression of MMP2 and MMP9 was downregulated remarkably post-Meq knockdown.The Meq knockdown could affect the main features of tumorous cells,including proliferation,apoptosis,and invasion,which indicates that the Meq gene plays a crucial role in promoting lymphomatous cell transformation.To elucidate the reason for gga-miR-130b downregulation in MDV-infected tissues,we analyzed differential methylation levels of upstream region of the gga-miR-130b-3p gene between MDV-infected tumorous and non-infected spleens.Around the upstream 1 kb of gga-miR-130b-3p gene,two amplicons were designed that covered 616 bp.There were 48 CpG sites in this region.CpG sites in this region presented higher methylation level in tumorous spleens compared with that in non-infected ones.There were eight CpG sites significantly hypermethylated in tumorous spleens.The expression level of three DNA methyltransferases including DNMT1,DNMT3a and DNMT3b increased and the expression level of Tet ten-eleven translocation protein 2 remarkably decreased in tumorous spleens.Hypermethylation in the upstream region of gga-miR-130b-3p gene might be a direct reason for its downregulation in MD tumorous tissues.Moreover,cell proliferation,migration as well as MMP2 and MMP9 expression were remarkably inhibited after gga-miR-130b-3p-agomir transfection.These results indicated that gga-miR-130b-3p plays an inhibitory role in lymphomatous cell transformation.The MSB1 cell proliferation and migration decreased post gga-miR-140-3p-agomir transfection.MMP2 expression was significantly downregulated at 24h,48h and 72 h;in addition,MMP9 expression was remarkably downregulated at 48h after gga-miR-140-3p-agomir transfection.In conclusion,gga-miR-140-3p which was downregulated in MDV-induced tumorous tissues affected features of tumor cells such as proliferation,migration and invasion,which indicates that this miRNA was involved in MD tumor transformation.In summary,this study demonstrated that gga-miR-219b,gga-miR-130b-3p and gga-miR-140-3p played an inhibitory role in chicken MD tumorigeneis through mediating proliferation,migration and invasion of MD tumor cells.BCL11B,as one target gene of gga-miR-219b,and Meq,as one oncogene of MDV,promoted MD tumor transformation.