| Osteosarcoma is one of the most common malignant tumors of bone,which used to appear mostly in adolescent.The malignant degree of osteosarcoma is high,it is not only easy to recur but also has high early metastatic rate,and the treatment is still one of the most challenging tasks in bone tumor.In the newly diagnosed patients,about 10% to 20% of the patients had distant metastasis,and 90% of them were lung metastases.Once the osteosarcoma was metastasized and relapsed,it often indicated that the prognosis of osteosarcoma was not good.The 5-year overall survival rate of osteosarcoma is 20-30%.At present,the treatment mode of osteosarcoma is "preoperative neoadjuvant chemotherapy and postoperative adjuvant chemotherapy".Neither the improvement of chemotherapy intensity,the change of combination of chemotherapeutic drugs,nor the addition of immunotherapy have made any further breakthrough in survival rate.The reason is that about 30-50% of patients with osteosarcoma still have uncontrollable lung metastasis.Therefore,the key to improve survival rate of osteosarcoma is to reduce the incidence of lung metastasis and find the molecular markers associated with lung metastasis.Integrin is a kind of transmembrane receptor that mediates the connection between cells and their external environment.It can recognize many kinds of extracellular matrix molecules such as fibrin/collagen and laminin and then cause the change of tumor cell adhesion.Preliminary results showed that the level of integrin ?4 in peripheral blood of osteosarcoma patients with lung metastasis and non-lung metastasis was not statistically different,but the patients with lung metastasis showed a higher trend than non-lung metastasis patients,but there was no significant difference between the patients with lung metastasis and the patients with non-lung metastasis.Whether the correlation between the two can be found by improving the sensitivity of detection methods has become a key problem to be solved.Exsome is a vesicle about 30-100 nm in diameter secreted by living cells,and it is a key medium for mediating local microenvironment and communication between distant microenvironment and extracellular matrix.It plays an important role in promoting the progression and metastasis of primary tumor.According to research reports,tumor exocrine integrin determines the organ specificity of tumor metastasis.Integrin ?4 plays an important role in lung metastasis.The expression level of integrin ?4 can be used to predict lung metastasis.The exosome of osteosarcoma cells can transport a variety of proteins called mi RNAs.Related signaling pathway molecules enhance tumor cell proliferation,invasion and metastasis by regulating tumor microenvironment.In conclusion,in order to study the role of exsome integrin ?4 in pulmonary metastasis of osteosarcoma and its related mechanism,the following studies were carried out in order to find the molecular markers of pulmonary metastasis of osteosarcoma.So as to provide a theoretical basis for clinical.Methods and Results:1.Clinical data analysis and detection of peripheral blood integrin ?4 and exosome integrin ?4(1)Clinical data analysis 82 cases of osteosarcoma patients hospitalized in Xijing Hospital,Xi'an Air Force military Medical University(formerly 4th military Medical University)from January 2014 to May 2017,including 48 males and 34 females aged 9-54years,were collected.The median age was 17 years old.The prognostic analysis showed that the factors influencing survival were staging,the number of pulmonary metastases and the treatment of lung metastases,in which staging and treatment of lung metastases were independent prognostic factors affecting survival.(2)ELISA was used to detect the levels of integrin ?4 in serum and exosome of patients with osteosarcoma and normal subjects.The levels of integrin ?4 and exsome integrin ?4 in patients with osteosarcoma were significantly higher than those in normal controls.Furthermore,the level of serum exsome integrin in osteosarcoma patients was significantly higher than that in serum integrin ?4.The serum secretions of osteosarcoma patients were extracted and separated by ultra-high speed gradient centrifugation(Osteosarcoma was divided into three groups: preoperative,no postoperative lung metastasis,postoperative lung metastasis).Then the morphology and size of the exsome were identified by projective electron microscope,and the existence of the characteristic molecular marker CD63?CD81 was identified by flow cytometry.Flow cytometry and Western-Blot were used to detect the serum levels of exsome integrin ?4 in the three groups.The results showed that the pulmonary metastasis group after operation > the preoperative group > the no pulmonary metastasis group after operation.It is suggested that exsome integrin ?4 is related to pulmonary metastasis of osteosarcoma.The analysis of patient data showed that exsome integrin ?4 was correlated with the stage of osteosarcoma patients(P = 0.01).It was correlated with lung metastasis(P=0.0018),and size of lung metastasis(p=0.0076).There was no significant correlation of patient's sex,tumor location,primary tumor size,operation or not,preoperative neoadjuvant chemotherapy or not and the efficacy of neoadjuvant chemotherapy.2.To analyze the biological function of exsome integrin ?4 in osteosarcoma cells.(1)A lentivirus vector containing overexpression and interference expression of integrin ?4 was constructed.The lentivirus was packaged and the virus titer was determined.After infection with osteosarcoma cell line MG-63,the lentivirus vector was constructed.The screening markers carried by the vector were used to screen the stable cell lines.Finally,Western-Blot and RT-PCR methods were used to identify the difference between integrin ?4 protein and m RNA expression after lentivirus transfection.(2)the control group was detected by scratch method and Transwell method.The changes of cell migration and invasiveness in exsome integrin ?4 overexpression group and exsome integrin ?4 interference expression group.The results showed that,compared with the control group,the exsome integrin ?4 overexpression group was treated with exsome integrin ?4 overexpression.Cell migration and invasion increased.(3)MMP-2 was detected by Western-Blot method in control group,exsome integrin ?4 overexpression group and exsome integrin ?4 interference expression group.The changes of MMP-9 protein expression showed that exsome integrin ?4 enhanced the expression of MMP-2 and MMP-9 protein compared with the control group.However,integrin ?4 decreased the expression of MMP-9 protein.(4)the model of tumor-bearing mice was established.The size and number of pulmonary metastases in tumor-bearing mice were detected after treatment with control group and interference secreting integrin ?4 group.The invasiveness of osteosarcoma cells in nude mice and the expression of MMP-2 and MMP-9 in nude mice were observed by HE staining and immunohistochemistry.The results showed that compared with the control group.The expression of integrin ?4 decreased the ability of invasion and metastasis of osteosarcoma cells,the number and size of metastatic foci were lower than that of the control group,and the expression of MMP-2 and MMP-9 protein decreased.3.Molecular mechanism of exocrine integrin ?4 in invasion and metastasis of osteosarcoma(1)The expression of Integin ?4 in osteosarcoma cells was determined by Western-blot method.The results showed that the expression level of Integrin ?4 did not change with the overexpression of Integrin ?4 and the total protein of ERK1/2 and phosphoric acid in osteosarcoma cells.There was no significant change in the expression of p38 MAPK in Integrin ?4 overexpression group compared with that in the control group.The results suggested that the ERK1/2 expression in MG63 cells of osteosarcoma was enhanced by Integrin ?4.On the contrary,the expression of p38 MAPK in osteosarcoma MG63 cells was increased.There was no significant change in the level of JNK/p38 MAPK after interference expression of Integrin ?4,while the total protein and phosphorylation level of ERK1/2 were lower than those of the control group.(2)To further confirm the above results,we use the 10umol/ml JNK inhibitor SP600125 p38 MAPK inhibitor SB203580 and ERK1/2 inhibitor PD98059 respectively with Integrin ?4 expression in MG63 cells by 60 min interference.The results show that the interference of Integrin ?4 decreased significantly after the invasion of MG63 cells after treatment with PD98059,Integrin ?4 the interference cells can significantly reduce the expression of MMP-2?MMP-9 to reduce the invasiveness of MG63 cells;while the SB203580 and SP600125 treated cells invasion found no significant changes.Therefore,the results suggest that Integrin ?4 mediated osteosarcoma cell invasion and metastasis by regulating ERK1/2 signal pathway.Conclusion:1.By analyzing the real world data of clinical osteosarcoma patients,found that the incidence of pulmonary metastasis in osteosarcoma was high,and lung metastasis seriously affected the survival of osteosarcoma,the prognostic factors included stage,number of metastatic foci and treatment of lung metastasis.Further multivariate analysis showed that staging and lung metastasis were independent factors affecting the prognosis of osteosarcoma patients.2.Exsome integrin ?4 plays an important role in the pulmonary metastasis of osteosarcoma,and there is a positive correlation between exsome integrin ?4 and pulmonary metastasis of osteosarcoma,which plays a guiding role in lung metastasis of osteosarcoma.3.The mechanism of ERK1/2 is involved in the process of osteosarcoma metastasis by exsome integrin ?4. |
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