JSRV Env Causes Malignant Transformation In Sheep Trophoblast Cells And Research On P53/AMPK/mTOR Signaling Pathway

Jaagsiekte sheep retrovirus(JSRV)is the pathogen of ovine pulmonary adenocarcinoma(OPA),which is an infectious lung cancer with malignant transformation of secreting epithelial cells in sheep lung.Some studies have shown that JSRV-env can act as an oncogene.The JSRV Env protein alone can transform mouse NIH3T3 cells,rat 208 F cells,chicken fibroblasts and canine epithelial cells,and can induce lung cancer in mice and sheep.Therefore,the envelope protein of JSRV Env has the rare characteristic of causing cell transformation and carcinogenesis,but the carcinogenic mechanism of Env is still unclear.In mammals,the mTOR(mammalian rapamycin target)protein kinase is the central node for nutrient and growth factor signaling.The most widespread tumor suppressor protein is p53,and p53 plays a key role in sensing genotoxicity and other stresses.However,there are few in-depth studies on the relationship between p53/AMPK/mTOR signaling pathway and the development of OPA tumor,and the further study on the relationship between cell transformation induced by JSRV Env.To further explore the function of ex JSRV enveiope protein,the eukaryotic expression plasmids pEGFP-C1/ex JSRV-env and pEGFP-C1/enJSRV-env preserved in our laboratory were transient transfected in sheep choriotrophoblast cells.The optimal transfection efficiency was detected by fluorescence microscopy 48 h after transfection.Shows that 10 ?L of LTX is the optimal transfection concentration that add to 2.5 ?g of eukaryotic expression plasmid.Detection of malignant transformation in sheep choriotrophoblast cells which were transfected with eukaryotic expression Plasmids pEGFP-C1/exJSRV-env,pEGFP-C1/enJSRV-env,and p EGFP-C1 empty plasmids by soft agar colony formation assay.The results showed that after transfection of pEGFP-C1/exJSRV-env plasmid,the cells grew in soft agar and colonies were generated,and the cell contact inhibition was disappeared;the transfection of pEGFP-C1/enJSRV-env plasmid,pEGFP-C1 empty plasmid and blank control group showed that could not produce colonies in soft agar.Detection of the effect of envelope protein expression on cell proliferation by plat clone assay.The results showed that the clone formation rate of sheep chorionic trophoblast cells transfected with pEGFP-C1/exJSRV-env was significantly higher than that of transfected pEGFP-C1/enJSRV-env Cells,p EGFP-C1 empty vector cells,and untransfected cells(P<0.01).The lung tissues of sheep with natural OPA infection and those of healthy sheep without OPA were used as negative controls.The Protein expression of p53/AMPK/mTOR signal pathway in the diseased lung tissues with OPA was detected by western blot method after extracting the total protein.The phosphorylation level of p53 and p-m TOR in OPA was significantly higher than that in healthy lung tissue.The phosphorylation level of p-AMPK in OPA was significantly lower than that in healthy lung tissue.The protein expression of p53/AMPK/mTOR signaling pathway in sheep choriotrophoblastic cells transformed by JSRV Env was detected by western blot assay.The results showed that p53 and p-mTOR were phosphorylated in JSRV Env transformed sheep chorionic trophoblastic cells.The phosphorylation level of p-AMPK in sheep choriotrophoblast cells was significantly lower than that in control cells.This study suggests that exJSRV Env expression can induce malignant transformation and cell proliferation of sheep choriotrophoblast cells,and preliminary studies on p53/AMPK/m TOR signaling pathways.It provides a theoretical basis for further study on the function of Jaagsiekte sheep retrovirus envelope protein,and provides a new idea for studying the tumorigenic mechanism of ovine pulmonary adenocarcinoma.