| The Wnt signaling pathway is a highly conserved signaling pathway that exists in the spine and invertebrates.It can be divided into two types:the canonical pathway that depends on ?-catenin and the non-canonical pathway that does not depend on ?-catenin.The Wnt signaling pathway plays an important role in embryonic development,tissue homeostasis,cell differentiation and cell proliferation.The abnormal activation of this pathway is closely related to the occurrence and development of tumors.At present,the research on the interaction between Wnt signaling pathway and disease is mostly focused on human and mouse diseases,and there is less on animal diseases.ALV-J is one of the diseases which can cause tumor in poultry.Currently,there are no vaccines and drugs for the prevention and control of the disease.Eradication is the major prevention and control method for ALV.There are few studies on the tumorigenic and immunosuppressive mechanisms of avian leukosis.This study focuses on the impact of ALV-J encoded protein on the Wnt signaling pathway,and then explores whether it participates in the regulation of the pathway and thus affects the cell growth cycle and tumor formation.At present,there are studies on the interaction between other virus-encoded proteins and the Wnt signaling pathway.The hepatitis C virus(HCV)1b gene core protein inhibits cell apoptosis and promotes the formation of hepatocarcinoma by activating the Wnt signaling pathway;the hepatitis B virus X protein induces abnormal activation of the Wnt signaling pathway,promotes cell proliferation,and participates in the formation of liver cancer.This study uses dual-luciferase reporter assay,real-time PCR and flow cytometry to study the influence of ALV-J encoded proteins on the signal pathway.And through activating the pathway by overexpression of ?-catenin in cells,we study the influence of the virus-encoded proteins on the signal pathway,which provides the foundation for further exploring the interaction mechanism between ALV-J and Wnt signaling pathway in poultry tumor diseases.1.The influence of ALV-J encoded proteins on Wnt signaling pathwayIn order to study the relationship between virus-encoded proteins and signal pathways,CEF cells were infected with whole virus.The Top flash activity and the expression level of related downstream genes of the signal pathway were detected by dual luciferase assay and real-time PCR to reflect the activation level of signal pathways in the virus-infected cells.The experimental results showed that compared with the low titer MOI=0.1 group,the Top flash activity of the MOI=1.0 group was significantly increased.And the expression levels of downstream genes LEF-1?TCF-4?AXIN??-catenin?c-myc and cyclinD1 were higher than low titer group.This showed that the whole virus MOI=1.0 could activate the signal pathway in CEF cells.Then the expression level of related genes in the downstream of the pathway was detected in CEF cells infected with different viral encoded proteins.The results showed that P27 could increase ?-catenin gene expression,an important transcription factor downstream of the pathway,which suggesting that the P27 protein might activate the signaling pathway.P2?P12?P15 and Gp37 down-regulated the expression of ?-catenin,which suggesting that they might inhibit the activation of signal pathway.The effect of virus-encoded proteins on the cell cycle detected by flow cytometry assay.The results showed that the virus-encoded proteins P2?P10?P12?P15?P27?P68?Gp3 7 and Gp85 could increase the proportion of cells in the S and G2 phases.These results revealed that P27 might increase the proportion of cells in the S and G2 phase by activating the Wnt signal pathway,thereby accelerating virus replication.2.The effect of virus-encoded proteins on Wnt signaling pathway in cells overexpressing ?-cateninTo further explore whether the virus-encoded proteins can promote the activation of the Wnt signaling pathway,the eukaryotic expression plasmid pCAGGS-?-catenin was constructed and transfected into CEF cells.The Top flash activity assay was used to confirm that overexpression of ?-catenin can activate the signal pathway.Then ?-catenin and the virus-encoded proteins were co-transfected into CEF cells.And the expression of related genes in the downstream of the pathway was detected by real-time PCR.The results showed that the expression of ?-catenin?LEF-1?TCF-4?AXIN and c-myc increased.The expression of cell cycle-related genes CCND1?CCNE1?CDK2 and CDK6 increased also.These results indicated that all the 10 proteins encoded by ALV-J could promote the activation of the signaling pathway in ?-catenin overexpressed cells.3.Construction of capsid protein P27 lentiviral vector and its expression in LMH cellsAccording to the results of the first two parts of this study,the virus-encoded protein P27 could up-regulate the expression of ?-catenin and increase the proportion of S-phase cells,which is beneficial to virus replication.Therefore,to further study the biological function of P27 in Wnt signal pathway,a lentiviral vector expressing the capsid protein P27 was constructed.The supernatant of the 293T cell packaging was infected with avian LMH cells.The expression of P27 was successfully detected at the gene and protein level,which indicating that the lentiviral vector expressing P27 was constructed successfully,and the assembled lentivirus can infect poultry-derived cells.This result provides a good tool for further research on the biological functions of P27. |
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