VCP Promoted Metastasis Of Osteosarcoma Through Autophagy Induction And Anoikis Inhibition

Osteosarcoma is a common malignant bone tumor in children and adolescents,with an important feature of early metastasis.For patients with metastatic disease,the current treatment effect is still awful.Pulmonary metastasis is the most important cause of death in patients with osteosarcoma.Therefore,in-depth study of the molecular mechanism of osteosarcoma metastasis,and seeking new prevention targets and intervention strategies is essential to improve the efficacy of patients with osteosarcoma.In previous studies,we have demonstrated that VCP is over-expressed in osteosarcoma tissue.And silencing of VCP expression in vitro by RNAi can down-regulate PI3K/AKT/NF-?? pathway and inhibit osteosarcoma cell invasion and migration.However,we also found that inhibition of the PI3K/Akt pathway does not completely reverse VCP-mediated osteosarcoma metastasis,suggesting that other mechanisms may be involved in VCP-mediated metastasis of osteosarcoma.Some researchers believe that autophagy can temporarily provide energy and necessary material for cells isolated from the matrix,delaying apoptosis.As a member of the adenosine triphosphate superfamily,VCP is closely related to energy metabolism and autophagy.We hypothesize that VCP is likely to enhance its "anoikis resistance" by inducing autophagy in osteosarcoma cells,thereby promoting osteosarcoma metastasis.In this study,human osteosarcoma tissue,osteosarcoma cell line and nude mice were used as research tools.The study was conducted in three parts:firstly,the evidence of VCP-induced autophagy was to be found from osteosarcoma tissues.Then,that VCP could induce autophagy,enhance anoikis resistance,promote osteosarcoma metastasis were to be confirmed in vitro,and the possible regulatory mechanisms were to be further explored.Finally,that VCP-induced autophagy could promote osteosarcoma growth and lung metastasis was further certified in nude mice.Part ? Expression of VCP in fresh osteosarcoma tissues and its relationship with autophagyObjective:To detect the expression of VCP in fresh osteosarcoma tissues,and to explore the relationship between VCP and autophagy.Methods:Fresh osteosarcoma tissues and paratumorous tissues were collected and the expression of VCP mRNA was detected by RT-PCR.The expression levels of VCP and autophagy related proteins were detected by Western Blot.Results:1.The expression level of VCPmRAN in fresh osteosarcoma tissues is significantly higher than that in paratumorous tissues.2.The expression levels of VCP,beclin-1 and LC3-?/? in fresh osteosarcoma tissues were all significantly higher than those in paratumorous tissues.Conclusions:1.VCP is highly expressed in fresh osteosarcoma tissues.2.The expression level of VCP is likely to be positively correlated with autophagy.Part ? VCP promoted metastasis of osteosarcoma through autophagy induction and anoikis inhibition via the ERK/NF-??/Beclin-1 signaling pathwayObjective:To investigate the role of VCP-induced autophagy in enhancing anoikis resistance and promoting osteosarcoma metastasis and possible mechanisms.Methods:The VCP down-expression lentiviral vector was constructed and transfected into 143B cells,and the expression level of VCP was changed by RNA interference technology.143B cells were cultured in suspension for 7 days and were treated with autophagy stimulator and inhibitors,as well as ERK inhibitor.The expression levels of VCP,autophagy and ERK/NF-?? signaling pathway-related proteins were detected.The ability of anoikis resistance,the migration and invasion for osteosarcoma cells was examined in vitro.Result:1.The expression level of VCP in osteosarcoma cells of RNA interference group was significantly lower than that of the control group.2.Compared with the control group,the ratio of LC3-?/? in the OS cells of the RNA interference group and the autophagy inhibitor treatment group was significantly down-regulated,while the ratio of LC3-?/? in the autophagy stimulator-treated OS cells was significantly up-regulated.3.After 7 days of suspension culture,the cell survival rate of RNA interference group was significantly lower than that of the control group.The cell survival rate of autophagy stimulator group was significantly higher than that of autophagy inhibitor group.In autophagy stimulator combined RNA interference group,the rate was significantly higher than that in the RNA interference group.4.Compared with the control group,the VCP down-regulated cells showed a significant decrease in migration rate and transmembrane cell number within 24 hours.There was no significant difference in the 24-hour migration rate and the number of transmembrane cells between cells treated with autophagy inhibitors or autophagy stimulators compared to the control group.5.When inhibiting VCP expression,the expression of ERK/NF-??p pathway protein was significantly down-regulated,as well as the autophagy-related protein.The expression levels of ERK,NF-??,Beclin-1 and LC3-?/? in ERK inhibitor treated group were significantly lower than those in the control group.Conclusion:1.High expression of VCP in osteosarcoma cells induces autophagy.2.Autophagy enhances the ability of anoikis resistance for osteosarcoma cells.3.High expression of VCP can promote the migration and invasion of osteosarcoma cells.4.The level of autophagy has little effect on cell invasion and migration ability.5.VCP induces autophagy by activating the ERK/NF-?? signaling pathway.Part ? VCP induced autophagy promotes osteosarcoma growth and metastasis in nude miceObjective:To investigate the role of VCP-induced autophagy in the promotion of osteosarcoma growth and metastasis in nude mice.Methods:Tumor-bearing nude mouse models were established and the expression level of VCP was altered by RNA interference technique.The autophagy level was changed by autophagy stimulator and inhibitor,and the growth and lung metastasis of osteosarcoma in nude mice were observed.Result:1.Subcutaneous and in situ tumor models in nude mice were successfully established.2.The tumor weight of the RNA interference group was significantly lower than that of the negative control group and the blank control group at 5 weeks.3.The tumor size of autophagy stimulator treated group,autophagy inhibitor treated group and blank control group was similar at 3 weeks.However,the lung metastasis rate of autophagy stimulator treated group was significantly higher than that of blank control group and autophagy inhibitor treated group.Conclusion:1.VCP promotes the growth of osteosarcoma in nude mice.2.Autophagy promotes lung metastasis in osteosarcoma of nude mice.