| Background and purpose:Osteosarcoma(OS)is the most common primary malignant bone tumor in children and adolescents.60%-70% of patients have an onset age of 10-25 years,and its incidence is about 4/1000000 per year worldwide.Osteosarcoma is not only easy to relapse but also has a high early metastasis rate because of its high invasiveness.In the newly diagnosed osteosarcoma patients,about 10% to 20% of patients have distant metastasis which 90% is lung metastasis--the main cause of death.Once osteosarcoma occurs metastasis and recurrence,it often indicates an extremely unfavorable prognosis and 5-year overall survival(OS)rate is merely 20% to 30%.At present,the widely accepted therapeutic model of osteosarcoma--preoperative neoadjuvant chemotherapy + tumor resection + postoperative adjuvant chemotherapy--is commonly used worldwide.In recent years,with the advancement of comprehensive treatments,the 5-year survival rate of osteosarcoma patients has notably increased,reaching more than 50%-68%,which has been greatly improved compared with the 1970 s.However,many patients with osteosarcoma still show resistance to currently available chemotherapy drugs,and then die from extensive metastasis and tumor recurrence,which is a major obstacle to successful treatment of patients with osteosarcoma.It is critical to develop targeted drugs to achieve precise treatment of osteosarcoma.Therefore,we need to explore key molecules and pathways in the occurrence,proliferation and metastasis of osteosarcoma,and provide new drug targets for the targeted treatment of osteosarcoma.Amphiphysin-1(AMPH-1)is a 128 k D endocytosis protein,which is rich in nerve endings and is involved in clathrin-mediated synaptic vesicle endocytosis and neuronal synaptic growth.Studies have shown that AMPH-1 plays an important role in the circulation of synaptic vesicles at nerve endings.At the same time,the low expression of AMPH-1 in neurons is related to neuronal degeneration and is involved in neuronal amyloidosis in patients with Alzheimer's disease.The yeast homologue of AMPH-1 promotes cell cycle switching,suggesting that AMPH-1 may play an important role in tumor progression.In recent years,AMPH-1 has been found to be low-expressed in lung and breast cancer specimens and is associated with poor patient prognosis.Studies have shown that AMPH-1 as a tumor suppressor gene can inhibit the occurrence,proliferation,migration and metastasis of lung cancer and breast cancer cells in vivo and in vitro.Therefore,AMPH-1 may be a key gene to effectively inhibit the occurrence and development of malignant tumors.The purpose of this study aimed to explore the following questions:(1)Detecting the expression level of AMPH-1 in osteosarcoma smaples and its relationship with the prognosis of patients with osteosarcoma;(2)Exploring the biological regulation of AMPH-1 in the progression of osteosarcoma in vitro and in vive;(3)Exploring the molecular mechanism by which AMPH-1 performs in biological functions in osteosarcoma.Method:(1)Osteosarcoma and osteofibrous dysplasia tissue were obtained in clinic,then immunohistochemical analysis revealed the results of the expression of AMPH-1;We clarify the relationship between AMPH-1 expression and prognosis of patients with osteosarcoma by Kaplan-Meier curve,and analyzed the relationship between AMPH-1 and clinicopathological characteristics of patients with osteosarcoma by chi-square test;(2)To establish a stable transfected osteosarcoma cell line by knockdown of AMPH-1,and explore possible effect of different expression of AMPH-1 on malignant behavior including proliferation,progression,and apoptosis;(3)To identify the significant role of AMPH-1 regulating the MEK/ERK signal pathway in malignant behavior of osteosarcoma by western blot experience;(4)To establish the xenograft mice model by subcutaneous injection of osteosarcoma cells and invest the significant role of knockdown of AMPH-1 in tumor growth in vivo.Results:(1)Immunohistochemical analysis revealed that AMPH-1 was downregulated in osteosarcoma tissues.Patients with low expression of AMPH-1 in osteosarcoma specimen significantly have poor overall survival.(2)The proliferation of osteosarcoma cells with knockdown of AMPH-1 is enhanced,and apoptosis is significantly reduced.The proportion of cells in G2 and S phases is significantly increased,and the proportion of cells in G1 phase is significantly reduced.The proportion of G2 and S phase cells was significantly increased,and the proportion of G1 phase cells significantly reduced.(3)The knockdown of AMPH-1 increased the expression level of MEK and ERK in osteosarcoma cell lines.(4)Nude mouse osteosarcoma model was constructed by subcutaneously injecting normal / knock-down osteosarcoma cells.The osteosarcoma in nude mice injected with osteosarcoma cells that knock down AMPH-1 is larger and heavier.The expression of phosphorylated ERK in tumor samples of the experimental group increased significantly.Conclusion:AMPH-1 is low expressed in osteosarcoma tissue,and patients with low expression of AMPH-1 have a poor prognosis.Cell experiments has confirmed that knockdown of AMPH-1 can promote the development of osteosarcoma cells in vitro and in vivo.Nude mice injected subcutaneously with knockdown AMPH-1 had larger tumors and heavier tumors.In terms of mechanism,we determined that AMPH-1 exerts its role by inhibiting the MEK / ERK signaling pathway,but more specific mechanisms need to be further clarified in the next experiments.In summary,our study indicates that AMPH-1 can inhibit the progression of osteosarcoma,and might provide a potential therapeutic target for osteosarcoma. |
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