USP3 Promotes Invasion And Metastasis Of Osteosarcoma By Regulating EPHA2 Homeostasis

Purpose:To explore the biological function and specific molecular regulation mechanism of USP3(ubiquitin-specific protease 3)in osteosarcoma.METHODS:The expression difference of USP3 in various tumors was analyzed by TIMER2.0 website(http://timer.comp-genomics.org/).WB(western blot)and RT-q PCR(Real-time quantitative PCR)techniques was used to detect the expression difference of USP3 in osteosarcoma tissue and adjacent tissue;ubiquitin-omics sequencing was adopted to find the downstream target protein of USP3;The TARGET children’s tumor database was used to analyze the relationship between the expression level of EPHA2 and the survival prognosis of osteosarcoma.Immunohistochemical staining scores were used to analyze the correlation between the expression levels of USP3 and EPHA2 and the clinical prognosis of osteosarcoma patients;The expression level of USP3 or EPHA2 in osteosarcoma cells was interfered by gene interference technology;In vitro,CCK-8,Migration,and Transwell experiments were respectively used to observe proliferation,migration and invasion activity of cells;Subcutaneous tumor formation in nude mice was used to detect the proliferation ability of osteosarcoma in vivo,and tumor cells were injected into the tail vein to establish a distant lung metastasis model to detect the invasion and metastasis of osteosarcoma;Mechanism analysis uses Co-IP(Co-Immunoprecipitation)and in vivo ubiquitination experiments to detect the ubiquitination levels of USP3 and EPHA2 and the interaction between the two.WB detection of the regulatory effect of USP3 on the PI3K/AKT signaling pathway.Results.1.USP3 was highly expressed in osteosarcoma and was assoriated with metastasis: Through bioinformatics analysis,it was found that USP3 was highly expressed in various tumors,but there was no data on the expression of USP3 in osteosarcoma.By detecting the difference in the expression of USP3 in fresh osteosarcoma tissue and paratumor tissue,it was found that the expression level of USP3 in osteosarcoma tissue was higher than that in paratumor tissue.By analyzing the relationship between the expression level of USP3 and clinical prognosis,it was found that the expression level of USP3 in the tumor tissue of patients with lung metastasis was higher than that of non-lung metastasis group,and the expression level of USP3 in advanced patients(Class III/IV)was higher than that in early(Class I/II)patients.2.USP3 promoted the proliferation,invasive metastatic ability of osteosarcoma cells in vivo and in vitro: After knocking down the expression of USP3 in the osteosarcoma cell line 143 B and HOS,the proliferation,migration and invasion of osteosarcoma cells were significantly inhibited;On the contrary,the proliferation,migration and invasion abilities of osteosarcoma cells were significantly promoted after overexpressing the expression of USP3.It was found that after down-regulating USP3 in vivo,the ability of subcutaneous tumor formation in nude mice was significantly reduced,and there were fewer distant lung metastases.3.USP3 promotes proliferation,invasion and metastasis of osteosarcoma by regulating EPHA2,and activating PI3K/AKT signaling pathway: Ubiquitination sequencing results show that after down-regulating the expression of USP3,there are116 proteins ubiquitin Increased levels.The top five are RAD18,EPHA2,ITG2 C,PFN1,USP5.Through bioinformatics analysis of the relationship between the single gene expression levels of these five genes and the survival and prognosis of osteosarcoma,it was found that only the expression level of EPHA2 was related to the survival prognosis of osteosarcoma patients.After down-regulating or up-regulating the expression of USP3 in osteosarcoma cells,the protein level of EPHA2 was correspondingly down-regulated or up-regulated,while the m RNA expression level of EPHA2 there was no significant change.Through the detection of Co-IP technology,it was found that EPHA2 and USP3 can specifically bind to each other.In addition,the protein degradation rate was traced by inhibiting protein synthesis with cycloheximide(CHX,cycloheximide);It was found that down-regulating USP3 could accelerate the degradation of EPHA2 protein.Conversely,up-regulation of USP3 inhibited the degradation of EPHA2 protein.By detecting the expression difference of EPHA2 in fresh osteosarcoma tissue and adjacent tissue,it was found that the expression level of EPHA2 in osteosarcoma tissue was higher than that in adjacent tissue.It was found that the expression level of EPHA2 was correlated with distant metastasis and clinical stage by Univariate analysis.After knocking down the expression of EPHA2 in 143 B and HOS cells,the proliferation,migration and invasion of osteosarcoma cells was significantly inhibited;On the contrary,the proliferation,migration and invasion of osteosarcoma cells was significantly promoted after overexpressing the expression of EPHA2 in 143 B and HOS cells.Overexpression of USP3 promoted the activation of PI3K/AKT signaling pathway;knocking down EPHA2 on this basis inhibited PI3K/AKT signaling pathway,and the proliferation,migration and invasion activities of osteosarcoma cells were also inhibited.Conclusion: 1.USP3 is significantly highly expressed in osteosarcoma,which is negatively associated with metastasis of patients;2.USP3 promotes invasion and metastasis of osteosarcoma by regulating EPHA2 protein homeostasis,and activating PI3K/AKT signaling pathway;3.USP3 might function as a potential therapeutical target for patients with osteosarcoma.