The Mechanism Of ALV-J Induced Tumor Formation In Chickens Regulated By Circular RNA Vav3

Subgroup J avian leukosis(AL-J) is the neoplastic and immunosuppressive disease induced by subgroup J avian leukosis virus(ALV-J).Recent reports showed that circular RNA(circRNA) plays an important role in the neoplastic diseases.Considering that up to now,there was no report regarding to ALV-J induced tumor formation regulated by circRNAs,hence,this study aims to explore the regulation mechanism of differentially expressed circRNAs in ALV-J induced tumor chickens to provide new theoretical basis for prevention and control of ALV-J.The first part: Screening of differentially expressed circRNAs in ALV-J infected chickensThe offspring in one day off(F3 generaiton)of ALV-J resistance chickens(F2 generation)and ALV-J susceptible chickens(F2 generation) were infected with ALV-J NX0101.Using RT-PCR and virus isolation two experiments to detect ALV-J infection in every week,after detection 20 weeks,three negative chickens in ALV-J resistant group and three positive plus tumor lesion chickens were chosen to conduct circ RNA sequencing.Total 5 kinds of circRNAs were identified in chickens including exonic,intronic,antisense,sense overlapping,intergenic,meanwhile,exonic circRNAs occupied the largest proportion of circRNAs which were identified in chickens,while intronic circRNAs occupied the smallest proportion.Comparing chicken circRNAs with those of mouse and humans,homology analysis results showed that the homologies of circRNAs in chickens compared with humans is 63.82%,while the homologies of circRNAs in chickens compared with mouse is 59.80%.Total 2808 differentially expressed circRNAs were identified in ALV-J induced tumor chickens and ALV-J resistance chickens.GO annotation of up-regulated circRNAs in ALV-J induced tumor chickens showed that those circRNAs were involved in molecular function,cellular component and biological process.KEGG signaling pathway analysis showed that those circRNAs were mainly involved in cysteine and methionine metabolism signaling pathway.Total 12 differentially expressed up-regulated circRNAs were also screened in ALV-J resistant group,indicating that those 12 differentially expressed up-regulated circRNAs may be the key genes for host against ALV-J infection.GO annotation analysis showed that those 12 differentially up-regulated circRNAs executed the function of molecular function regulator,channel regulator activity and so on.They were located in the cellular component of transferase complex and so on,and they were involved in the regulation of biological process,such as the reactive oxygen species.KEGG signaling pathway showed that those 12 differentially up-regulated circRNAs were involved in RNA transport,FOXO signaling pathway and m TOR signaling pathway.The second part: Validation of oncogenic circ RNA circ-Vav3 sponges with gga-miR-375Under the basis of screened oncogenic circRNAs,bioinformatics softwares were used to predict the possible adsorbed miRNAs for those circRNAs.Previous study in our lab showed that gga-miR-375 plays a key role in tumor formation induced by ALV-J,after we predicted the possible adsorbent miRNAs,we found that a circ RNA named circ-Vav3 had six seed sequences which were matched with gga-miR-375,hence,RIP,RNA-pulldown and RNA-FISH three experiments were conducted to confirm their interaction.Those three experiments showed that circ-Vav3 did sponge with gga-miR-375.RNA-FISH result further showed that circ-Vav3 located in the cytoplasm,gga-miR-375 located in both cytoplasm and nuclear,while circ-Vav3 sponges with gga-miR-375 in the cytoplasm.The third part: The mechanism research of ALV-J induced tumor formation regulated by circ-Vav3/gga-miR-375/YAP1 axisUnder the basis of circ-Vav3 spongs with gga-miR-375,we continue to research the down stream genes of circ-Vav3/gga-miR-375 and to explore whether they affect tumor formation.Dual luciferase reporter was used to confirm that YAP1 was the target gene of gga-miR-375.The overexpression of gga-miR-375 could inhibit the protein expression level of YAP1.We further confirmed that YAP1 overexperssion could promote cell migration of DF-1 and induce EMT through up-regulation mesenchymal cell markers Fibronectin and N-cadherin,down-regulation the epithelial cell marker E-cadherin.Co-overexpression of circ-Vav3 and gga-miR-375 in DF-1 cells could up-regulate the transcription level and protein level of YAP1,Fibronectin and N-cadherin,while down-regulate the transcription level and protein level of E-cadherin.For further validation the accuracy of previous results,ALV-J NX0101 was used to infect DF-1 cells,results showed that ALV-J NX0101 could also up-regulate the transcription level and protein level of YAP1,Fibronectin and N-cadherin,while down-regulate the transcription level and protein level of E-cadherin.Lastly,clinical ALV-J induced tumor livers and normal livers were collected to conduct RT-q PCR for further detection those core genes expression level trend of circ-Vav3,gga-miR-375,YAP1,Fibronectin,N-cadherin and E-cadherin,and results also had the same expression trend like above.Hence,we conclude that circ-Vav3/gga-miR-375/YAP1 axis through induction EMT by up-regulation mesenchymal cell markers Fibronectin and N-cadherin,down-regulation epithelial cell marker E-cadherin to promote tumor formation in chickens.In conclusion,this study is the first to prove that circ-Vav3/gga-miR-375/YAP1 axis promote tumor formation in ALV-J infected chickens by inducing EMT through up-regulation the transcription level and protein level of Fibronectin and N-cadherin,down-regulation the transcription level and protein level of E-cadherin.