The Mechanism Of MD Tumor Induced By Meq Protein Through PI3K/Akt Signaling Pathway

Marek's disease(MD)caused by Marek's disease virus(MDV)is a T lymphoproliferative disease in chickens.MDV-induced tumors and immunosuppression induced vaccines failure increased mortality of infected chickens.Since discovered in 1907,MD has been plaguing the global poultry industry for a long time and has caused huge economic losses.Although the vaccination is an important measure to prevent and control MD,the virulence of MDV is increasing because of the immune pressure and the evolution of the virus genome.In recent years,the outbreaks of MD are still spreading in China and even in the world.Therefore,it is urgent to further study the molecular mechanism of MDV in pathogenesis and tumorigenesis,so as to provide an important theoretical basis for elucidating the molecular mechanism and vaccine development of MDV.In order to screen the tumor-associated signaling pathways activated by MDV-infected host cells,the isolated MDV strain LZ1309 was used to infect chicken embryo fibroblasts,then the MDV-infected group and the control group were subjected to transcriptome sequencing,GO function enrichment analysis and Pathway Enrichment analysis.GO functional analysis showed that signal transduction,membrane transport,transmembrane transport and other molecular functions of gene expression of CEF cell were abnormally after MDV infection.Pathway enrichment analysis showed that the expression of genes were upregulated which related to PI3 K,p53,MAPK,Fox O and other signaling pathways in MDV-infected CEF cells,and the PI3K/Akt signaling pathway was closely related to other major signaling pathways(Signaling pathway of P53,MAPK,Fox O and so on)activated by LZ1309-infected CEF cells.The MDV-encoded Meq protein is a key tumour-inducing protein and the amino acid sequence contains multiple PXXP motifs that interact with the p85 subunit of the PI3 K protein in the PI3K/Akt signaling pathway.The above study found that virulent MDV strain infection could activate the PI3K/Akt pathway.It is speculated that MDV may participate in the MD-tumor formation through activation of PI3K/Akt signaling pathway by Meq protein.To confirm this hypothesis,the interaction of MDV virus protein Meq with PI3 K protein was studied by immunoprecipitation and laser confocal technique.Co-IP results confirmed that the Meq protein interacts with the p85? and p85? subunits,respectively.The eukaryotic plasmids expressing Meq were co-transfected with eukaryotic plasmids expressing p85? and p85? subunits separately,and the confocal microscopy scanning showed that Meq protein could co-localize with both p85? and p85? subunits in the cells.The eukaryotic plasmid expressing Meq protein was transfected into DF1 cells and found to activate the PI3K/Akt signaling pathway.Compared with the parental strain,the Meq protein-deleted strain significantly reduced the activation of the PI3K/Akt signaling pathway.The experimental results showed that MDV's Meq protein could interact with the p85 subunit of PI3 K protein to activate the PI3K/Akt signaling pathway.PI3K/Akt signaling pathway plays an important role in many oncology diseases.To study whether this pathway is involved in the regulation of MDV-induced tumorigenesis,MSB-1 tumor cells were treated with the PI3 K specific inhibitor LY294002 and through animal experiments to observe whether the PI3K/Akt signaling pathway was activated in tumor tissues.The results showed that LY294002 could inhibit the replication of MSB-1 cells,promote apoptosis,and arrest cell cycle at G1 phase.The PI3K/Akt signaling pathway related proteins were detected by immunohistochemistry and pathological sections of LZ1309-challenged SPF chickens at 60 dpi.The results of immunohistochemical and pathological sections showed that the activation of phosphorylated Akt and GSK-3? was observed in tumor tissues.Western blot confirmed that the phosphorylated Akt and GSK-3? were highly expressed in tumor tissues.The results showed that PI3K/Akt signaling pathway could be activated in MDV-induced visceral tumors in chickens,indicating that this pathway play an important role in the mechanism of MD tumorigenesis.In summary,this study found that MDV Meq protein could interact with the p85 subunit of PI3 K protein to activate the PI3K/Akt signaling pathway in the host cell,and verified that this pathway involved in MDV-induced tumor formation.This study provided a reference for the further study of the molecular mechanism of MDV interaction with host cells and the tumorigenesis mechanism of MDV.