Deubiquitinating Enzyme USP10 Promotes Osteosarcoma Epithelial-Mesenchymal Transition And Metastasis By Stabilizing YAP1

Background and Objective:Osteosarcoma is a malignant tumor originating from bone tissue that is common in children and adolescents.It is characterized by high malignancy,rapid growth,and easy metastasis.At present,the treatment of osteosarcoma is still a major challenge.USP10 can deubiquitinate the proteins in the cytoplasm,thereby participating in the regulation of cell growth,migration and apoptosis,which in turn affects the occurrence and development of tumor cells.As one of the YAP isoforms in the important downstream effector of Hippo signaling pathway,YAP1 plays an important role in tumor cell proliferation,apoptosis,autophagy and distant metastasis.Studies have shown that both USP10 and YAP1 are involved in the regulation of epithelialmesenchymal transition and distant metastasis of tumor cells,thereby affecting the occurrence and development of tumors.However,the roles and mechanisms of the two in osteosarcoma are rarely studied.This study mainly explored the expression and correlation of USP10 and YAP1 in osteosarcoma,and investigated the effect of USP10 in osteosarcoma on epithelial-mesenchymal transition and tumor metastasis,in order to provide a new theory for clinical targeted therapy of osteosarcoma patients and basis.Methods:1.The expression levels of USP10 in osteosarcoma tissues were analyzed by immunohistochemistry,real-time fluorescence quantification and western blotting,and the expression levels of USP10 in normal osteoblasts and osteosarcoma cell lines were detected by real-time fluorescence quantitative and western blotting.2.The constructed shUSP10 plasmid was transferred into osteosarcoma cells,and the plasmid with the most obvious down-regulation of shUSP10 level was selected for the experiment.The expression levels of epithelial-mesenchymal transition-related proteins were observed by Western blotting after infection of osteosarcoma cell lines with shUSP10 plasmid,and then the effects of cell invasion and migration were observed by scratch healing assay and Transwell assay.Finally,the osteosarcoma cell lines stably transfected with sh NC/143 B and shUSP10/143 B were injected subcutaneously on both sides of the buttocks of nude mice,respectively,to construct an experimental model of tumor formation in nude mice to observe the tumor formation.The osteosarcoma cell lines stably transfected with sh NC/143 B and shUSP10/143 B were injected into the tail vein to observe the occurrence of lung metastasis of pulmonary osteosarcoma in nude mice.3.To analyze the expression level of YAP1 in osteosarcoma tissue and adjacent tissue by immunohistochemistry,real-time fluorescence quantitative and Western blotting,and analyze the correlation between USP10 and YAP1 in osteosarcoma tissue.The expression of YAP1 was further analyzed by altering the expression of USP10 in osteosarcoma cells.Then,YAP1 was up-regulated in shUSP10 osteosarcoma cell line and the expression of YAP1 was inhibited in over-expressed USP10 cell line.The expression of EMT-related proteins was detected by Western blot and the ability of cell invasion and migration was observed by Transwell assay.Finally,the Co-IP method was used to detect whether USP10 could directly bind to YAP1 in osteosarcoma cell lines.4.The effect of USP10 on the expression level of YAP1 was detected by Western blot under the action of proteasome inhibitor(MG132).Results:1.Immunohistochemical results of osteosarcoma and its adjacent tissues showed that USP10 was highly expressed in72.41%(21/29)of osteosarcoma tissues,but only increased in 17.24%(5/29)of adjacent tissues(p <0.01).q RT-PCR and Western blot detection confirmed that USP10 was highly expressed in osteosarcoma(p<0.01).In normal osteoblasts and osteosarcoma cell lines,U2-OS,143 b,MG63,and Saos-2 cell lines were detected by q RT-PCR and Western blot detection.The expression of USP10 in U2-OS and 143 b was significantly higher than that in normal osteoblasts(p<0.01).2.After transfection of U2-OS and 143 b cell lines with shUSP10 plasmid in vitro experiments,q RT-PCR and Western blot detection showed that EMT-related proteins were changed,and the expression level of E-Cadherin protein was significantly increased(p<0.05),while the expression levels of N-Cadherin protein and Vimentin protein were significantly decreased(p < 0.05).The wound healing test and Transwell test confirmed that the invasion and migration of osteosarcoma cells were significantly inhibited(p < 0.01).In vivo tumorigenesis experiments also showed that the size of subcutaneous tumorigenic tumors in nude mice and the number of lung metastases after injection by tail vein were significantly reduced in osteosarcoma cells compared with the control group(p<0.05).The above results confirmed that shUSP10 can inhibit the occurrence of EMT and distant metastasis of osteosarcoma cells.3.After down-regulating the expression of USP10 in osteosarcoma by Western blot analysis,it was found that the expression of YAP1 also decreased;on the contrary,when overexpressing USP10,YAP1 increased significantly.By immunohistochemical analysis of osteosarcoma tissue and adjacent tissues,we found that the expression level of YAP1 was significantly higher than that in adjacent tissues,and YAP1 expression was positively correlated with the expression of USP10.Meanwhile,this study also found that the EMT of osteosarcoma could be rescued by up-regulating the expression of YAP1 in the shUSP10 cell line,and the invasive and metastatic ability of osteosarcoma was enhanced.It can inhibit the EMT of osteosarcoma,and the invasive and metastatic ability of osteosarcoma is also reduced.We confirmed that USP10 and YAP1 can directly bind and interact with each other by Co-IP.4.We also confirmed that the expression of YAP1 increased with the prolongation of the action time of proteasome inhibitor MG132 in osteosarcoma cells.After changing the expression of USP10 in osteosarcoma cells,it was found that the expression of YAP1 did not change significantly compared with the control group after treatment with MG132,and the degradation rate of YAP1 protein in osteosarcoma cells decreased significantly.Finally,our study confirmed that USP10 can promote osteosarcoma epithelial-mesenchymal transition and metastasis by stabilizing the expression of YAP1 by deubiquitination.Conclusion:We observed that USP10 can promote osteosarcoma EMT and distant metastasis by stabilizing the expression of YAP1 in vivo and in vitro.Our research results provide a new theoretical basis for EMT and metastasis of osteosarcoma,and provide a new target for clinical targeted therapy of osteosarcoma.