| Background:Osteosarcoma is the most common primary bone malignancy in children and adolescents with high recurrence and metastasis rates.The preferred treatment options were neoadjuvant chemotherapy,tumor resection and postoperative adjuvant chemotherapy.Over the past few decades,multiple clinical trials have shown no significant improvement in patient outcomes by altering the dosage of chemotherapy drugs or combining them with other new drugs.Chemotherapy resistance is an important problem that restricts the improvement of prognosis in patients with osteosarcoma.Forkhead box M1(FOXM1)is a proliferation-related transcription factor that is closely related to cell proliferation,self-renewal and tumorigenesis.It maintains tumor characteristics mainly by regulating the expression of target genes at the transcriptional level.Studies have shown that FOXM1 plays an vital role in the progression of osteosarcoma.Previous studies by our team have found that FOXM1 is associated with the proliferation,migration and chemotherapy resistance of osteosarcoma cells,suggesting that FOXM1 may be an important regulatory factor for chemotherapy resistance and malignant progression of osteosarcoma,but its specific molecular mechanism remains unclear.Wnt/β-catenin signaling pathway is a classical pathway that regulates embryonic development,cell differentiation,proliferation and homeostasis of adult stem cells.Studies have shown that β-catenin,as a biomarker of osteosarcoma,can promote the metastasis of osteosarcoma to the lung.In addition,it has been found that the Wnt/β-catenin pathway can promote the invasion and migration of osteosarcoma cells in recurrent osteosarcoma.Our previous results showed that Wnt signaling pathway may be inactivated in the early stage of osteosarcoma,promoting normal bone differentiation and development and limiting tumor growth.However,in the advanced stage of osteosarcoma,the expression of tumor stem cell-like features is enhanced by up-regulating the Wnt signaling pathway,and the malignant progression of tumor cells such as chemotherapy resistance and metastasis is promoted.Therefore,this study focused on whether FOXM1 regulates the Wnt/β-catenin signaling pathway to promote chemotherapy resistance in osteosarcoma.Methods:human osteosarcoma cell lines HOS and U2 OS were routinely cultured,and stable drug-resistant cells HOS/R and U2OS/R were screened with increasing cisplatin concentration(maintenance concentration 1μg/ml).Lentivirus was used to construct stable overexpressing FOXM1 cells(HOS/FOXM1,U2OS/FOXM1).The effects of chemotherapy resistance and overexpression of FOXM1 on proliferative ability and susceptibility to drug resistance of osteosarcoma cells were determined by CCK8,plate clonal formation and EDU assay.The protein expressions of FOXM1 is the downstream target genes of Wnt/β-catenin: Cyclin D1,C-myc,and cancer stem cells markers: OCT4,nanog were detected by WB assay.The effects of overexpression of FOXM1 and chemotherapeutic resistance on the growth rate and sensitivity of osteosarcoma cells were examined in BALB/c-nude mouse transplanted tumor models.The interaction between FOXM1 and β-catenin was detected by immunoprecipitation.Graph Pad Prism software(version 8.0.2)and SPSS software(version 16.0)were used for statistical analysis.Student’s-t test and one-way ANOVA method were used to compare the two groups,and P<0.05 was considered statistically significant.Results :FOXM1 in drug-resistant cells was significantly higher than that in parent cells by WB method.CCK8 experiment showed that under the cisplatin concentration gradient,the proliferation ability of FOXM1 overexpressed cells was higher than that of empty cells,and the difference was statistically significant(P<0.05).Plate cloning and EDU experiments showed that the proliferation ability of FOXM1 overexpressed cells was significantly higher than that of empty cells,and the difference was statistically significant(P<0.05).The total protein and nuclear protein of β-catenin were significantly increased in drug-resistant cells and overexpressing FOXM1 cells by WB assay,and Cyclin D1 and C-myc were also significantly increased in drug-resistant cells and overexpressing FOXM1 cells.Total protein and nuclear protein of β-catenin decreased after treatment with FOXM1 inhibitor RCM-1(10μg/m L).In the overexpressed FOXM1 cells and drug-resistant cells,plate cloning and EDU experiments showed that the proliferation ability of the cells treated with RCM-1(10μg/m L)and cisplatin(0.5μg/ml)was significantly decreased compared with that treated with cisplatin alone.Tumor formation experiment in nude mice showed that the tumor growth rate of Fox M1-overexpressed cells was higher than that of empty cells.In drug-resistant cells and Fox M1-overexpressed cells,the growth rate of the combination of RCM-1 and cisplatin was significantly lower than that of cisplatin alone,and the difference was statistically significant(P<0.05).The results of WB showed that the tumor stem cell marker genes OCT4 and Nanog were significantly increased in drug-resistant cells and overexpressing FOXM1 cells,while the tumor stem cell marker genes OCT4 and Nanog were significantly decreased in drug-resistant cells and overexpressing FOXM1 cells after treatment with the inhibitor RCM-1.Immunocoprecipitation showed a direct interaction between β-catenin and FOXM1.Conclusions: In this study,FOXM1 expression was significantly increased in cisplatin resistant cells,and overexpression of FOXM1 increased cisplatin resistance in osteosarcoma cells.Overexpression of FOXM1 or use of FOXM1 inhibitor RCM-1 can up-regulate or down-regulate the expression of Wnt/β-catenin signaling pathway and its downstream target genes(Cyclin D1 and C-myc).The use of FOXM1 inhibitor in combination with chemotherapeutic drug resistance was reversed in cell and nude mouse tumor formation experiments.FOXM1 overexpression promoted the stem cell expression of osteosarcoma tumor.Further studies found that FOXM1 and β-catenin directly interact;We speculate that FOXM1 may promote chemotherapy resistance to osteosarcoma by activating the Wnt/β-catenin pathway to up-regulate the expression of tumor stem cell characteristics. |
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