Research On The Relationship Between Wnt/β-catenin Signaling Pathway And MDV Replication

Marek’s disease（MD）is a malignant T-lymphocyte tumor disease caused by Marek’s disease virus（MDV）,which mainly infects chickens and turkeys.It is easy to cause mixed infection of other viruses and bacteria,which causing huge economic losses to the poultry industry around the world.Although the currently widely used CVI988 vaccine can provide better immune protection and greatly reduce the mortality of chickens,it still cannot prevent the spread and infection of MDV virus particles.At the same time,under the selection of vaccine immune pressure,the virulence of MDV is also with continuous evolution,MDV infection still occurs sporadically in CVI988 immune-protected flocks.So far,the tumorigenic mechanism of MDV infection is still unclear,and how the virus induces tumorigenesis and development by interacting with host cells has not been elucidated.The Wnt/β-catenin signaling pathway is highly conserved during evolution and is ubiquitous in various animals.It is a key signaling pathway in the growth process of the body,which can control cell polarity,growth and differentiation,etc.,and is involved in cancer also.Its abnormal activation is closely related to the occurrence and development of tumors.As a virus that can cause tumors,the relationship between Wnt/β-catenin signaling pathway and MDV has not been reported yet.This study mainly focused on the relationship between the Wnt/β-catenin signaling pathway and the replication and proliferation of MDV,and in-depth study of the influence of virus-encoded proteins on the signaling pathway.The experimental results of this study provide a new theoretical basis for elucidating the mechanism of tumor induced by MDV infection.1.The effect of MDV replication on Wnt/β-catenin signaling pathwayIn order to study the effect of MDV on the Wnt/β-catenin signaling pathway,this study carried out both in vivo and in vitro studies.In vivo,after infecting SPF chickens with MDV RB1B strain,RNA was extracted from the main organs（brain,bursa,thymus,spleen,kidney,liver）that the virus invaded,and reverse transcription was performed for quantitative real-time PCR（Quantitative Real-time PCR,qRT-PCR）detection,the results showed that the Wnt/β-catenin signaling pathway related factors were all upregulated,and with the gradual increase of virus infection in the body,the expression of related genes in the pathway was up-regulated more and more significantly.There is a close relationship between the β-catenin signaling pathways,and MD V infection can significantly activate the response of the Wnt/β-catenin signaling pathway in the host.Based on the results of in vivo experiments,after infecting chicken embryonic fibroblasts（CEFs）with MDV RB1B strain in vitro,in addition to extracting cellular RNA to detect gene expression levels,laser confocal technology was also used to detect MDV-infected CEF cells.The localization of β-catenin.Consistent with the results of in vivo experiments,β-catenin in the cytoplasm entered the nucleus at 72 h of MDV infection,suggesting that the Wnt/β-catenin signaling pathway was activated,and the results of genetic testing were consistent with it.The above experimental results show that MDV RB1B strain infection can activate the Wnt/β-catenin signaling pathway in vivo and in vitro,but how does MDV activate the Wnt/β-catenin signaling pathway activation pathway,and the activation of the pathway will affect the replication of MDV.Further research is needed to explore the effect of proliferation.2.The effect of the activation state of Wnt/β-catenin signaling pathway on the replication and proliferation of MDVIn order to study the effect of Wnt/β-catenin signaling pathway on viral replication,this study first treated MDV-infected cells with the signaling pathway-specific activator Lithium Chloride（LiCl）,and collected cellular RNA and total protein by indirect Immunofluorescence（IFA）,Western blot（WB）,qRT-PCR,plaque count,plaque area measurement and other techniques were used to detect the replication and proliferation of MDV after activation of the pathway.The results show that,in terms of viral gene expression level,protein expression level and plaque formation,LiCl activation of the pathway can significantly inhibit the replication and proliferation of the virus,and this inhibitory effect is not only dose-dependent and time-dependent.,and also acts in every link of virus infection.At the same time,cytokines related to innate immunity and inflammation in cells were also detected,and the results showed that the expressions of interferon-related genes and inflammation-related genes were significantly downregulated.At the same time,the signaling pathway-specific inhibitor iCRT14 was also used in this study.By treating MDV-infected CEF cells with iCRT14 and collecting their RNA,qRT-PCR and plaque formation results showed that MDV replication and proliferation were promoted when the pathway was inhibited.At the same time,an experiment was designed to first use the pathway activator LiCl to infect the virus,and then use iCRT14 to inhibit the pathway to investigate whether the inhibitory effect on MDV after pathway activation is reversible.The expression level of MDV gene and the results of plaque formation showed that iCRT14 alleviated the inhibitory effect on MDV after the activation of Wnt/β-catenin signaling pathway,indicating that the activation of Wnt/β-catenin signaling pathway inhibited the replication and proliferation of MDV.The above experimental results show that when the pathway is activated,it has an inhibitory effect on the replication and proliferation of cells.When the pathway is inhibited,it has a promoting effect on the replication and proliferation of MDV.3.Effects of MDV-encoded proteins on Wnt/β-catenin signaling pathwayIn order to explore how MDV activates the Wnt/β-catenin signaling pathway,this study constructed pCAGGS-Meq,pCAGGS-pp38,pCAGGS-vIL8,pCAGGS-LORF9 cloned plasmids of four virus-encoded protein genes,transfected into CEF and LMH cells,qRT-PCR,dual luciferase reporter assay and WB assay were performed to detect the effect of virus-encoded protein on Wnt/β-catenin signaling pathway.The results showed that the four virus-encoded proteins all had an activating effect on signaling pathways and up-regulated the expression of downstream cancer-related genes（such as C-Myc,mmp7）,among which Meq and pp38,the main tumorigenic genes of MDV,had the most up-regulation of pathway-related genes,significantly.As genes closely related to the generation and transformation of tumors,pp38 and Meq play an important role in the process of virus-infected hosts causing tumors and pathogenesis.After transfecting them into CEF,the cells were treated with iCRT14,a signaling pathwayspecific inhibitor.The results showed that the activation of the virus-encoded protein on the pathway was inhibited by iCRT14,which indicating that the virus-encoded protein was specific for the activation of the Wnt/β-catenin signaling pathway.The experimental results of this study provide a new theoretical basis for elucidating the mechanism of MDV infection-induced tumor.