Effect Of Ambra1 On Chemotherapy Sensitivity Of Osteosarcoma U2OS Cells

Background:Osteosarcoma is the most common primary malignant bone tumor in children and adolescents,accounting for 30%of primary malignant bone tumors.The 5-year survival rates for children(0-14 years)and adolescents(15-19 years)were 69.6%and 65.7%,respectively,while metastatic patients were only 20-30%.In the past 20-30 years,there has been no breakthrough in the treatment of OS.At present,the treatment of OS is mainly chemotherapy and surgery.Chemotherapy plays an important role in the treatment of OS.However,even with the standard use of neoadjuvant chemotherapy,the prognosis for OS remains unsatisfactory.Among them,chemotherapy resistance is the main reason of poor prognosis.Although a large number of studies have been conducted on chemotherapeutic resistance,the mechanism of chemotherapeutic resistance is still unclear.Rosenberg first reported on cisplatin's anticancer properties in 1973.Currently,cisplatin is an essential and important member of the first-line chemotherapy for OS,and its mechanism of action is relatively complex.The most clearly studied pattern is the cross-linking of DNA between and within the chains,which activates the apoptosis signal transduction pathway by forming dna-platinum adjunction,leading to cell death.There is also experimental evidence that other mechanisms,such as the production of reactive oxygen species and the activation of inflammatory pathways,may also contribute to the induction of apoptosis.Ambra1,also known as autophagy/beclin-1 modulator 1,is an essentially highly disordered conserved binding protein in vertebrates and is part of the autophagy signaling network.Ambra1 is associated with embryonic development,neurological disorders,and tumors.Previous studies have shown that Ambra1 can cause resistance to chemotherapy drugs in colon,ovarian,prostate,neuroblastoma and breast cancer.The role of Ambra1 in the tolerance of OS is unclear.In this study,we found that Ambra1 can reduce the sensitivity of cisplatin-mediated OS cells to U20S.Objective:1.To investigate the effect of cisplatin intervention on the expression of Ambra1 in OS cells U20S;2.To investigate the effect of the expression level of Ambra1 on the sensitivity of cisplatin to OS cells U20S.Methods:1.The expression level of Ambra1 mRNA was detected by microarray in the cancer and adjacent tissues of 3 patients with OS.2.The expression levels of Ambra1 mRNA in human osteoblasts hFOB1.19 and 3 human OS cells(U20S,MG63 and Saos2)were detected by RT-qPCR.3.Different concentrations of cisplatin were used to intervene U20S cells.The apoptosis rate of U20S cells at different concentrations was detected by flow cytometry.The expression of Ambra1 mRNA in U20S cells at different concentrations was determined by RT-qPCR.4.U20S cell lines with overexpression and interference of Ambra1 were constructed by lentivirus transfection,and the expression of Ambra1 mRNA was detected by RT-qPCR to confirm the success of cell model construction.5.The caspase 3 activity and the expression of LC3B were detected using the same concentration of cisplatin as the intervention for intransfection and transfection of U20SResults:1.Ambra1 was expressed in the cancer tissues and adjacent tissues of 3 patients with OS,and its expression in the cancer tissues was lower than that in the adjacent tissues.2.Ambra1 was expressed in human osteoblast hFOB1.19 and 3 human OS cells(U20S,MG63 and Saos2).The expression of Ambra1 in Saos2 was higher than HfOB,the expression in MG63 was basically consistent with hFOB 1.19,and the expression in U20S was lower than hFOB 1.19.3.With the increase of cisplatin concentration,the apoptosis rate gradually increased,and the expression of Ambra1 also gradually increased.4.Cisplatin at the same concentration interfered with the cells,and the overexpression group of Ambra1 showed decreased caspase 3 activity and increased LC3B expression.In contrast,the caspase-3 activity increased and LC3B expression decreased in the Ambra1 interference group.Conclusion:Cisplatin intervention in OS cells U20S will lead to the increase of Ambra1 expression,and the high expression of Ambra1 will reduce the sensitivity of human OS cell line U20S to cisplatin,which is related to the autophagy mediated by Ambra1.