| Background and purposes:Osteosarcoma(OS)is one of the most lethal malignancies of children and adolescents,mainly because of its propensity for pulmonary metastasis in the early stage of disease.There was significant improvement in the five-year survival rate of patients without metastasis(55-80%)in traditional surgery,radiotherapy and chemotherapy.However,in recent decades,due to the high recurrence rate of osteosarcoma,multi-drug resistance to chemotherapy and early lung metastasis,the prognosis of patients with osteosarcoma has not been significantly improved.Therefore,it is urgently finding a new molecular markers and therapeutic targets related to osteosarcoma.In our previous study,we found that Aurora-B is highly expressed in osteosarcoma tissue and is closely related to lung metastasis.However,its potential role in the process of lung metastasis of osteosarcoma and its specific cytological and molecular mechanisms are still unclear.As a double-edged sword,autophagy plays an important role in tumor metastasis.However,the role of Aurora-B in autophagy and lung metastasis in osteosarcoma and its mechanism have not been reported.The completion of this study further clarifies the role and possible regulatory mechanism of Aurora-B in osteosarcoma metastasis and provides new strategies and interventions for the prevention and treatment of clinical osteosarcoma.Methods:1.The expression and distribution of Aurora-B in tumor tissues correlated with the survival prognosis and the expression of autophagy-related proteins in patients with osteosarcoma: The GEPIA database was used to analyze the expression and distribution of Aurora-B gene in different tumor tissues,including sarcoma.Survival and prognosis of patients with sarcoma in the database were obtained to analyze the relationship between Aurora-B gene expression and survival and prognosis of patients with sarcoma.The tumor tissues of 69 patients with osteosarcoma were collected clinically,and immunohistochemical staining was used to detect the expression and distribution of Aurora-B and autophagy-related proteins.The relationship between Aurora-B expression and tumor size,metastasis,pathological stage and prognosis of patients with osteosarcoma was analyzed based on patients’ hospitalization information and follow-up after hospital release.2.The effect of Aurora-B knockdown on autophagy,invasion,and metastasis in osteosarcoma cells: The expression of Aurora-B protein in osteosarcoma cell lines was knockdown by lent-interfering virus vector(LV-sh RNA).The formation of autophagosomes in osteosarcoma cell lines was observed by LC3 double-labeled fluorescence and transmission electron microscopy.The expression of autophagy related proteins and autophagic flux were detected by Western blot.The Transwell invasion and migration experiment and Wound healing scratch experiment were used to detect the invasion and migration ability of osteosarcoma cells,and Western blot was used to detect metal matrix proteases to explore the molecular mechanisms related to the migration ability of osteosarcoma cells.Chloroquine blocked activated autophagy and further examined the role of Aurora-B-induced autophagy knockdown in invasion and migration of osteosarcoma.3.The role of Aurora-B Knockdown in invasion and migration in osteosarcoma cells by inhibiting mTOR signaling activated autophagy: Western blot was used to detect the expression of mTOR pathway-related proteins,and mTOR activator was used to further test the effect of Aurora-B knockdown on autophagy.Transwell invasion,migration test and Wound healing scratch test were used to detect the invasion and migration ability of osteosarcoma cells.Use mTOR activator to reactivate the mTOR signaling pathway,and further test the role of the mTOR signaling pathway in autophagy,invasion,and migration of osteosarcoma.4.The effect of Aurora-B knockdown-mediated autophagy on lung metastasis of osteosarcoma in vivo: The orthotopic nude mouse model of osteosarcoma was constructed using the nude mouse in situ tumorigenesis method,and the nude mouse models were divided into 4: control group,AZD2811 group,AZD2811+3BDO group,AZD2811+CQ group.Dissecting and collecting tumor tissues in situ,using immunohistochemistry to detect the expression and distribution of Aurora-B and phosphorylated mTOR in each group of tumors,collecting lung tissues,using the small animal in vivo imaging technology,HE staining,stereomicroscope,etc.to observe the original Lung metastasis in nude mice.Results:1.The GEPIA database analysis shows that compared with the corresponding adjacent tissues,Aurora-B is highly expressed in most tumor tissues,among which there are 29 kinds of relatively high expression tumors.A separate analysis of the expression of Aurora-B in sarcoma tissues and corresponding adjacent tissues found that the expression of Aurora-B in sarcoma tissues was significantly higher than that in adjacent tissues(P<0.05).Among the 262 patients with sarcoma in the database,Kaplan-Meier analysis found that the expression of Aurora-B was significantly related to the survival and prognosis of patients with sarcoma,and the high expression of Aurora-B predicted a worse overall survival rate(P=0.0041)and disease-free survival.Rate(P=0.00078).In the tumor tissues of 69 patients with osteosarcoma,the expression of Aurora-B was negatively correlated with the Enneking stage(P=0.031)and the expression of autophagy-related protein LC3B(P=0.016)in patients with osteosarcoma.Kaplan-Meier analysis found that the high expression of Aurora-B protein was negatively correlated with the overall survival rate(P=0.0022)and metastasis-free survival rate(P=0.0107).2.The expression of Aurora-B protein and m RNA in osteosarcoma cell line was successfully knocked down by using lentiviral vectors.LC3 double-labeled fluorescence and transmission electron microscopy showed that knocking down the expression of Aurora-B protein in osteosarcoma cells significantly increased intracellular autophagosomes and autophagolysosomes.Western blot analysis showed that the ratio of autophagy-related protein LC3Ⅱ/I increased,while the expression of P62 decreased significantly.Inhibiting the expression of Aurora-B protein significantly increased the formation of autophagic flux in tumor cells.In order to explore the effect of autophagy induced by Aurora-B knockdown on invasion and migration in osteosarcoma cell lines,we used Transwell invasion and migration experiments and Wound healing scratch experiments to find that knockdown of Aurora-B protein expression significantly reduced osteosarcoma Cell invasion and migration ability.Using CQ to block activated autophagy can significantly reverse the inhibited migration and invasion ability of osteosarcoma cells.Western blot analysis found that the knockdown of Aurora-B protein in osteosarcoma cells significantly inhibited the expression of MMP2 protein,and the use of CQ could significantly restore the expression of MMP2.3.Western blot analysis found that knockdown of Aurora-B significantly inhibited the activity and expression of the mTOR signaling pathway,and the application of mTOR agonists could significantly restore the activity of mTOR and the expression of autophagy-related protein P62.In order to further explore the effect of mTOR on autophagy and invasion and migration in osteosarcoma cell lines,Transwell invasion,migration assay and Wound Healing scratch assay were used to show that the reactivation of mTOR significantly reverse the inhibition of Aurora-B knockdown on the migration and invasion of osteosarcoma cells.4.Tumor-bearing experiments in nude mice showed that the application of AZD2811,a specific inhibitor of Aurora-B,successfully inhibited the expression of Aurora-B protein in nude mice with osteosarcoma,and significantly reduced the expression of p-mTOR.The combination of AZD2811 and mTOR agonist 3BDO was used to reactivate mTOR activity in the nude mouse models.Bioluminescent imaging analysis,HE staining and in-situ microscopy were used to observe lung metastases in nude mice.It was found that the use of AZD2811 effectively reduced the number of lung metastases in the nude mouse model of osteosarcoma.The combined application of AZD2811 and 3BDO or autophagy inhibitor CQ treatment effectively restored the number of lung metastases reduced by Aurora-B inhibition.Conclusion:Aurora-B expression is abnormally elevated in a variety of tumors including sarcoma and is closely related to the overall survival rate of patients with osteosarcoma,the expression of autophagy-related proteins,Enneking staging,and distant metastasis.Knockdown of Aurora-B protein expression in osteosarcoma cells can activate autophagy and inhibit the lung metastasis ability of osteosarcoma through the mTOR signaling pathway. |
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