| Avian leukosis(Avian Leukosis,AL)is a collective term for a variety of tumorous diseases in poultry caused by Avian Leukosis Virus(ALV)and Avian Sarcoma Virus(RSV).Avian leukosisleukosis virus belongs to the family Retroviridae,a genus of retroviruses.Avian leukosisleukosis causes death,elimination of diseased birds,multiple tissues and multiple types of tumors.In recent years,the research on the pathogenic mechanism of avian leukosis virus has been a hotspot of the disease.Scientists at home and abroad have carried out a lot of research around the pathogenic and tumorigenic mechanism of avian leukosis virus infection and already obtained valuable scientific research progress.However,the mechanism of avian leukosis virus infection and pathogenesis is still poorly understood,especially how the virus uses intracellular signaling pathways to regulate the occurrence of cellular inflammation,cell biological changes,cause immune suppression,and eventually lead to tumor formation is still unknown.Recent studies have reported that infections with HIV,EBV,Hepatitis B virus,Hepatitis C virus,and Kaposi's sarcoma-associated herpes can cause abnormal activation of the Wnt signaling pathway,eventually leading to the occurrence of cancer tumors.The research field of Wnt signaling pathway in poultry started late,and some components of the Wnt signaling pathway in poultry tissue have been discovered and cloned recently.It has been reported that activation of the Wnt/?-catenin signaling pathway is necessary for the proliferation of primordial germ cells in poultry.Growth retardation of poultry caused by avian leukosis virus infection is caused by downregulation of the Wnt/?-catenin signaling pathway.However,the relationship between the canonical Wnt signaling pathway and the avian leukosis virus subgroup J replication is unknown.In this study,real-time PCR,Western blot,dual lucifase reporting system and other technical methods,as well as activators and inhibitors of the signaling pathway,were used to focus on the correlation between the canonical Wnt signaling pathway of GSK/?-catenin and replication of avian leukosis virus of subgroup J from two aspects of gene transcription and protein expression,which preliminarily revealed the effect of Wit signaling pathway on replication of avian leukosis virus,providing a new scientific basis for the comprehensive explanation of the pathogenic mechanism of avian leukosis virus.1.Activation of the canonical Wnt/?-catenin signaling pathway increases the replication of avian leukosis virus subgroup JIn order to study the relationship between the Wnt signaling pathway and the pathogenicity of ALV-J infection,the cell model sensitive to the Wnt signaling pathway was first identified as CEF cells.Then after activation of the Wnt signaling pathway by signaling pathway activator GSK3 inhibitor X,signaling pathway related molecules Expression,the expression of avian leukosis virus-encoded proteins,and the expression of related cellular inflammatory factors and antiviral genes were detected.The results showed that GSK3 inhibitor X can inhibit the activity of GSK-3?,and activate the expression of downstream genes such as ?-catenin,LEF1,TCF4,and Axin2 of Wnt signaling pathway.The expression level of virus-encoded proteins increased significantly,and virus proliferation significantly accelerated also.But these changes could be disturbed by the signaling pathway inhibitor iCRT14.The expressions of cytokines IL-1?,IL-6,interferon-stimulating factor OASL,ISG12-2 and antiviral factor viperin all decreased in varying degrees.The results of cell cycle experiments showed that after the cells were treated with the signaling pathway activator GSK3 inhibitor X,the proportion of S-phase cells increased significantly.Based on these results,activation of the canonical Wnt/?-catenin signaling pathway in CEF cells is beneficial to ALV-J replication and proliferation,which might be related to the regulation of the cell cycle by the Wnt signaling pathway.2.Inhibition of the canonical Wnt/?-catenin signaling pathway inhibits the replication of avian leukosis virus subgroup JIn order to investigate the effect of inhibiting the canonical Wnt/?-catenin signaling pathway on virus replication,this study treated CEF cells with the signaling pathway specific inhibitor iCRT14 to observe its effect on ALV-J replication.The experimental results showed that iCRT14 could significantly inhibit ALV-J replication in CEF as dose-dependent manner.When cells were treated with iCRT14,the expressions of ?-catenin,LEF1,TCF4,CyclinDl,C-myc,and Axin in the downstream of the Wnt pathway were also significantly reduced.The expression of cytokines IL-1? and interferon-stimulating factor OASL decreased significantly,while the expression of IL-6 increased significantly.At the same time,the cell growth cycle related genes CDK6,CCNE1,and CDK2 all showed a downward trend with the increase of the dose of iCRT14,and the cell growth rate became slower.In addition,the study also used siRNA to interfere with ?-catenin expression to inhibit signal pathway activation,and virus expression was also suppressed.These results indicate that iCRT14 inhibits the canonical Wnt signaling pathway and significantly inhibits the replication of the ALV-J in CEF cells,which might also be related to the regulation of the cell cycle by the Wnt signaling pathway.3.Effects of Wnt signaling pathway inhibitors on ALV-J replication in different cell typesPrevious reported studies indicated that the Wnt signaling pathway might exhibit different biological responses in different cell types.So this study also selected chicken liver cancer cell line LMH and chicken embryonic kidney cell CEK to study the effects of Wnt signaling pathway inhibitor iCRT14 on the replication of ALV-J.The results revealed that ALV-J could infect LMH and CEK cells.After treatment with iCRT14 inhibitors,the expression of ?-catenin and CyclinDl genes in the signaling pathway was significantly reduced,and ALV-J replication was also suppressed.This result is consistent with the results of CEF cells,which further indicating that inhibition of the canonical Wnt signaling pathway hinders the replication of ALV-J.The specific mechanism needs to be clarified in future study. |
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