Deubiquitination Enzyme USP10 Promotes Autophagy And Proliferation Of Osteosarcoma By Up-regulating ULK1 Expression

Research Background:Osteosarcoma is one of the most common malignant bone tumors and most commonly affecting children and adolescents.Osteosarcoma tends to occur in the long diaphyseal region,such as the distal femur and proximal tibia.Osteosarcoma has a low incidence but is characterized by highly malignant growth and metastasis.At present,the main clinical treatment of osteosarcoma is surgical resection of the tumor combined with drug chemotherapy,which has a low cure rate.More and more evidence show that effective chemotherapy drugs are crucial for the treatment and prognosis of osteosarcoma,and the exploration and development of efficient molecular targeted drugs is imminent.Ubiquitination is the process by which ubiquitin molecules in cells bind to specific proteins,and deubiquitination is the process by which ubiquitin molecules are removed from proteins.The balance between ubiquitination and deubiquitination plays an important role in maintaining protein stability,subcellular localization and signal transduction.Deubiquitinase inhibitors have been found to inhibit the growth and metastasis of osteosarcoma cells,so they are promising drug candidates for the treatment of osteosarcoma.USP10(ubiquitin-specific peptidase 10)is a member of the deubiquitination enzyme family that regulates cell growth,apoptosis,and DNA damage repair.USP10 can act as a tumor suppressor gene to inhibit the proliferation and invasion of cancer cells.In addition,USP10 may also act as an oncogenic gene and play a promoting role in some tumors.USP10 has a complex regulatory role in autophagy,which can promote or inhibit autophagy.ULK1(Unc-51-like kinase 1),an autophagy related kinase,is an important regulatory factor in the autophagy pathway and is crucial for the initiation of autophagy.Although both USP10 and ULK1 are associated with autophagy,there are few studies on their roles and related mechanisms in the development of osteosarcoma.The purpose of this study was to investigate the effects of USP10 and ULK1 on autophagy and proliferation of osteosarcoma,and the mechanisms between them.Objective:The purpose of this study was to clarify the effects of USP10 on autophagy and proliferation of osteosarcoma cells,and to explore the regulatory effect of USP10 on ULK1 and its molecular mechanism,so as to provide innovative theoretical basis for the study of malignant progression of osteosarcoma and a new target for clinical treatment of osteosarcoma.Methods:1.The mRNA and protein expression levels of USP10 in osteosarcoma cells and normal osteoblasts were detected by qRT-PCR and Western Blotting.2.Osteosarcoma cell lines with stable low or high expression of USP10 were established.Western Blotting was used to detect the expression of autophagy related markers(LC3B-Ⅱ and p62),autophagy levels were detected by transmission electron microscopy,and cell proliferation was detected by plate cloning assay.3.The USP10 expression level of osteosarcoma cells was changed.The protein expression level of ULK1 was detected by Western Blotting,and the mRNA expression level of ULK1 was detected by qRT-PCR.4.The expression levels of USP10 and ULK1 in osteosarcoma cells were changed at the same time.The expression of autophagy related markers(LC3B-Ⅱ,p62)was detected by Western Blotting,the autophagy level was detected by transmission electron microscopy,and the proliferation of cells was detected by plate cloning.5.Constructing 143 B osteosarcoma cell line stably transfected with shNC,shUSP10 and shUSP10+ULK1.The above three cell lines were used to construct a human osteosarcoma nude mouse model,and the tumor formation of nude mice was observed to determine the effects of USP10 and ULK1 on the growth of osteosarcoma cells.Results:1.The protein and mRNA expression levels of USP10 in osteosarcoma cells were significantly higher than those in normal osteoblasts.2.Overexpression of USP10 significantly enhanced autophagy and cell proliferation in osteosarcoma cells.In contrast,USP10 knockdown significantly inhibited autophagy levels and cell proliferation ability in osteosarcoma cells.3.Overexpression of USP10 significantly increased the protein and mRNA expression levels of ULK1 in osteosarcoma cells.In contrast,knockdown of USP10 significantly decreased ULK1 protein and mRNA expression levels in osteosarcoma cells.4.ULK1 overexpression promoted autophagy and proliferation of osteosarcoma cells and reversed the decreased autophagy level and proliferation ability of osteosarcoma cells caused by USP10 knockdown.5.When the expression of USP10 in osteosarcoma cells is down-regulated,the volume and weight of transplanted tumor in nude mice are significantly decreased,and this phenomenon can be reversed by ULK1 overexpression.Conclusion:In vitro and in vivo experiments showed that USP10 promoted autophagy and proliferation of osteosarcoma by increasing ULK1 mRNA and protein expression.Our findings suggest that USP10 may be a novel target for targeted therapy of osteosarcoma,providing a new direction and strategy for the treatment of osteosarcoma.