Studies On The Involvement Of Autophagy In Tumorigenesis Induced By Jaagsiekte Sheep Retrovirus Envelope

The envelope(Env)of Jaagsiekte sheep retrovirus(JSRV)has the ability to transform cultured cells in vitro and induce tumorigenesis in vivo,as an obligate parasitic retrovirus.However,how JSRV invasion breaks the cell balance and eventually leads to transformation is still unclear.Therefore,this thesis aims to explore the molecular mechanism of JSRV tumorigenesis from the perspective of cell dynamic balance.Autophagy is an important process to resist external stimuli and maintain normal physiological functions in cells.At present,there are no reports on the relationships between JSRV Env and cell autophagy.Therefore,the purpose of this paper is to explore the changes of autophagy in JSRV Env transformed cells,and the main experiments are as follows:1.The RNA-seq analysis was performed on naturally infected OPA lung tissue.The results show that there are 2,143 differentially expressed transcripts(adjust p<0.05)in OPA lung tissue,mainly enriched in 16 biological function modules.These functional modules are regulated by autophagy,and highly correlated with signaling pathways regulated by autophagy,such as PI3K-Akt-mTOR,MAPK signal pathways.The above results indicates that autophagy is may be involved in the tumorigenic process of JSRV.2.JSRV env was successfully recombined into vector pCMV-dR8.91,packaging and purification.The multiplicity of infection(MOI)gradient test confirms that the most suitable MOI for STCs is 5,and the most suitable MOI for infection of NIH 3T3 cells is 30.The above results indicate that JSRV env overexpression lentivirus is successfully constructed and successfully infected STCs and NIH 3T3 cells.3.JSRV env lentivirus infects STCs and NIH 3T3 cells for 72h.Cell proliferation,migration and invasion ability are significantly enhanced(p<0.05),and obvious colonies could be formed in soft agar within 10d.Transformed cells injected into nude mice for 28 d can form obvious subcutaneous tumors(STCs:1.94±0.83cm3;NIH3T3:3.24±0.47cm3).The above results indicate that STCs and NIH 3T3 cells infected by JSRV env lentivirus are transformed.4.The expressions of Beclin-1 and p62 are significantly down-regulated(p<0.05)and the expression of LC3 is down-regulated in OPA lung tissue and JSRV Env transformed cells.The autophagosomes is significantly reduced in JSRV Env transformed cells(p<0.05).Immunofluorescence co-localization of OPA lung tissue and protein immunoprecipitation test of JSRV Env transformed cells shown that there is an interaction between JSRV Env and Beclin-1.The above results indicate that the autophagy homeostasis and the number of autophagosomes are reduced in JSRV Env transformed cells,and there is a evidence of interaction between JSRV Env and Beclin-1.5.LC3 II turnover test results show that when lysosomal inhibitor(Pepstatin A)is used to inhibit the fusion of autophagosomes and lysosomes,the autophagosome membrane protein LC3 II shows a large accumulation in a short period of time in JSRV Env-transformed cells.The results of the p62 degradation test show that the autophagy inner membrane mosaic protein p62 is largely degraded with the prolonged transfection time(0-72h).The above results indicates that there is complete autophagy circulation in JSRV Env transformed cells.6.In the nude mice tumorigenesis model(STCs:1.17±0.06 cm3;NIH 3T3:1.23 ±0.01 cm3),3-MA(2mg/kg/d)and RAPA(1mg/kg/d)were given for 28d.Compared with the control group,the tumor volume and weight of the 3-MA group and RAPA group were significantly reduced(p<0.05).In JSRV Env tumors formed by STCs,the positive signal of E-cadherin in the 3-MA group was significantly greater than that of the control group(p<0.05),and the positive signal of Vimentin was significantly less than that of the control group(p<0.05).There was no significant difference between E-cadherin and Vimentin positive signals in RAPA group compared with control group.It shows that RAPA as an autophagy activator and mTOR inhibitor,its impact on tumor progression not only depends on autophagy regulation,but also may be related to mTOR signal transduction inhibition.And 3-MA inhibits tumor progression by reducing cell autophagy homeostasis and affecting cell transformation.The information of differentially expressed transcripts in the lung tissue of OPA can provides a research basis data for screening of OPA potential biological markers.In addition,the JSRV env overexpression lentivirus,and JSRV Env transformed cells and JSRV Env subcutaneous xenograft models in nude mice in this study can provide efficient and stable research tools for further revealing the molecular pathogenic mechanism of JSRV Env.Importantly,this experiment explores the corresponding changes and related roles of cellular autophagy in the process of cell transformation induced by JSRV Env,which can provide new ideas for the prevention and control of OPA in the future.