Modulation Of Autophagy In ExJSRV-env-NM-transformed Cells Through The Akt/mTOR And MAPK Signaling Pathways

Jaagsiekte sheep retrovirus(JSRV),which is genus Betaretrovirus belongs to family Retroviridae,subfamily Orthoretrovirinae.Jaagsiekte sheep retrovirus is the etiological agent of ovine pulmonary adenocarcinoma(OPA),a contagious neoplasm in sheep.Three signaling pathways were reported to mediate in JSRV-Env transformed cells:PI3K/Akt,MAPK and RON/Hyal2 pathway.It was shown that autophagy was suppressed in different types of cancers.Cellular autophagic activity was inversely associated with malignancy.But there were no relative reports about the relationship between autophagy formation and the development of OPA tumors,as well as the transformation in JSRV-Env induced cells.It attracted our interest whether JSRV-Env associated autophagy,modulating by which signaling pathways in OPA tumor cells and JSRV-Env transfected cells.In this study,we detected the activities of PI3K/Akt/mTOR and MAPK signaling pathways,as well as the levels of autophagy in OPA lung tissues and JSRV-Env transformed NIH3T3 and sheep trophoblast cell line by monitoring the expressions of Beclin-1 and LC3-? in vitro.Then we detected that autophagy was modulated by which signaling pathways in OPA lung tissues and JSRV-Env transfected NIH3T3 and sheep trophoblast cell line.These studies indicated that the biomechanism of JSRV-Env transformation was complexity.We established a basement of the study about the relationship between autophagy formation and the development of OPA tumors,as well as JSRV-Env transfected cells.1.All OPA and the control lung tissues were fixed in 4%formalin and embedded in paraffin wax.In the immunohistochemical(IHC)study,EGFR,Akt/mTOR and MAPK pathway were detected the location where they expressed.The results showed that EGFR,p-MEK1/2,p-ERK1/2,p-p38,p-JNK,p-Akt and p-mTOR were strongly phosphorylated in both cytoplasmic and nuclear compartments in OPA lung tumor tissues.The activation of Akt/mTOR and MAPK pathways of tissues in-80(?)by results showed that the phosphorylation levels of Akt/mTOR and MAPK pathways in OPA lung were significant higher than that in normal lung.2.Total RNA from lung tissues and transformed NIH3T3 cells was extracted and qPCR for the Beclin1 and LC3 genes were performed according to conventional methods.The results showed that The Real-time Quantitative analysis of Beclinl and LC3 transcripts confirmed that Beclinl and LC3 activation prominently decreased in the OPA lung tissues,in comparison to the control sheep lung tissues.In the immunohistochemical(IHC)study,Beclin-1 and LC3 were detected the location where they expressed.The results showed that Beclin-1 and LC3 expressed in both tumor cells in OPA lung and epithelial cells and stromal cells in normal lung.The exspression of Beclin-1 and LC3 ?/?of tissues in-80 ? were detected by western blot.The results showed that the protein levels of Beclin-1 and LC3 ?/? in OPA lung were significant lower than that in normal lung.3.Recombinant expression plasmid pcDNA4/myc-His/exJSRV-env was transfected into NIH3T3 cells and sheep trophoblast cells.500 ng DNA mixed LTX 2 ?L,3 ?L,4 ?L,5 ?L,4 ?L,6 ?L,8 ?L and 10 ?L,48 h after transfection,the optimal transfection efficiency and the expression of EGFR were detected by RT-PCR and western blot analysis.The results showed that the way that 500 ng DNA mixed 4 ?L LTX was the optimal transfection efficiency.The expression of EGFR in transfected pcDNA4/myc-His/exJSRV-env NIH3T3 cells and sheep trophoblast cells was significant higher than the normal cells.4.The expression of EGFR,p-MEK1/2,p-ERK1/2,p-p38,p-JNK,p-Akt and p-mTOR in pcDNA4/myc-His/exJSRV-env transfected NIH3T3 cells and sheep trophoblast cells was detected by western blot.The results showed that the phosphorylation levels of EGFR,p-MEK1/2,p-ERX1/2,p-p38,p-JNK,p-Akt and p-mTOR in pcDNA4/myc-His/exJSRV-env transfected NIH3T3 cells and sheep trophoblast cells were significant higher than that in normal cells.5.The expression of Beclin-1 and LC3 ?/? in pcDNA4/myc-His/exJSRV-env transfected NIH3T3 cells and sheep trophoblast cells was detected by qPCR and western blot analysis.The results showed that the expression of Beclin-1 and LC3 ?/? in pcDNA4/myc-His/exJSRV-env transfected NIH3T3 cells was significant lower than normal cells.The results showed that the expression of Beclin-1 in pcDNA4/myc-His/exJSRV-env transfected sheep trophoblast cells was significant lower than normal cells and the expression of LC3 ?/? in pcDNA4/myc-His/exJSRV-env transfected sheep trophoblast cells was significant higher than normal cells.6.The levels of mTOR,JNK,ERK1/2 and p38 MAPK in NIH3T3 and sheep trophoblast cells which were treated with Rapamycin,SP 600125,PD98059 and SB203580(0 ?mol/L,1 ?mol/L,5 ?mol/L,10 ?mol/L,20 ?mol/L and 50 ?mol/L)for 24 h,48 h,72 h were analyzed by western blot.The results showed that the inhibition efficiency in 20?mol/L for 48 h was optimum.The expression of Beclin-1 and LC3 ?/? was detected by qPCR and western blot analysis when the Akt/mTOR and MAPK pathways were blocked.The results showed that the expression of Beclin-1 and LC3 ?/? was significant higher than that in normal transfected cells when the Akt/mTOR and MAPK pathways were blocked.This study indicated that JSRV Env activated Akt/mTOR and MAPK pathways and inhibited the expression of Beclin-1 and LC3 ?/? through the two pathways.