The Mechanism Of FOXM1-mediated Autophagy In Methotrexate Resistance Of Osteosarcoma Cells

Objective:Osteosarcoma is more common in children and adolescents,with high malignancy and poor prognosis.Surgical resection of the tumor followed by neoadjuvant chemotherapy is the standard treatment for osteosarcoma.Cisplatin,doxorubicin and methotrexate（MTX）are the first-line chemotherapy drugs for osteosarcoma.Chemotherapy resistance caused by long-term chemotherapy,especially high-dose MTX,seriously affects the prognosis of osteosarcoma patients.Therefore,finding new therapeutic targets and exploring the intrinsic mechanisms leading to chemotherapy resistance are the keys to improve the problem of chemotherapy resistance in osteosarcoma.Autophagy is an intracellular process that degrades and removes damaged organelles and misfolded proteins,thereby maintaining cellular homeostasis.Studies have found that autophagy plays an important role in the chemoresistance of tumor cells,and inhibiting autophagy is an important strategy to overcome the chemoresistance of tumor cells.Forkhead box M1（FOXM1）is a transcription factor related to cell proliferation,which is widely involved in the proliferation,invasion and migration of tumor cells and other biological traits.Studies have shown that FOXM1 is highly expressed in osteosarcoma cells,but its role and mechanism in the resistance of osteosarcoma cells to MTX remain unclear.The aim of this study is to investigate the role of FOXM1 in the process of MTX resistance in osteosarcoma cells and further explore the molecular mechanism of FOXM1-induced MTX resistance in osteosarcoma cells.Methods:In this study,two osteosarcoma cell lines,U-2OS,143B and osteoblast h FOB1.19,were used to explore the role and molecular mechanism of FOXM1 in osteosarcoma MTX resistance.This study was carried out as follows:1.The expression of FOXM1 in osteosarcoma tissues and cells was detected by immunohistochemistry and Western blot.The relationship between FOXM1 expression and overall survival rate of osteosarcoma patients was analyzed by TARGET database.2.R package was used to analyze the expression of FOXM1 in MTX-resistant and MTX-sensitive cells in GEO database;The osteosarcoma MTX resistant cells were established by the concentration gradient shock method,and the IC₅₀ value of osteosarcoma MTX resistant cells was detected by CCK-8 method.The expression of FOXM1 protein in MTX-resistant cells and MTX-sensitive cells was detected by Western blot.3.Western blot and RT-q PCR were used to detect the expression levels of FOXM1 protein and m RNA in osteosarcoma cells after MTX treatment.4.The results of Western blot and RT-q PCR showed that the expression levels of FOXM1protein and m RNA were significantly down-regulated and up-regulated in FOXM1knockdown and FOXM1 overexpression osteosarcoma cells.5.CCK-8 assay was used to detect the proliferation ability of OS cells with overexpression or knockdown of FOXM1 after MTX chemotherapy.Flow cytometry was used to detect the apoptosis rate of osteosarcoma cells with overexpression or knockdown of FOXM1after MTX chemotherapy.Western blot was used to detect the expression levels of Cleaved-caspase3 and Bax proteins in osteosarcoma cells with or without FOXM1 overexpression after MTX chemotherapy.6.Western blot and immunofluorescence assay were used to detect the expression of LC3B protein in osteosarcoma cells with or without FOXM1 overexpression after MTX chemotherapy.7.The apoptosis of FOXM1-overexpressing osteosarcoma cells treated with MTX and CQ was detected by flow cytometry.8.Co-immunoprecipitation assay was used to detect the interaction between FOXM1 and HMMR.The expression of HMMR protein in FOXM1-knockdown osteosarcoma cells was detected by Western blot.9.Flow cytometry was used to detect the effect of HMMR overexpression on the apoptosis rate of FOXM1-knockdown osteosarcoma cells after MTX chemotherapy.10.Western blot was used to detect the expression of ATG7,a downstream protein of FOXM1 involved in the regulation of autophagy,and the effects of FOXM1 and HMMR on the expression of ATG7.11.The effect of FOXM1 knockdown on the sensitivity of osteosarcoma cells to MTX in vivo was verified by subcutaneous tumor formation in nude mice.12.TUNEL fluorescence staining and LC3B immunofluorescence staining were used to detect cell apoptosis and LC3B expression in tumor tissues of mice in each group.Results:1.The results of immunohistochemistry showed that FOXM1 was highly expressed in tumor tissues compared with adjacent tissues;Western blot results showed that the expression of FOXM1 protein in osteosarcoma cells was significantly higher than that in h FOB1.19 cells.TARGET database analysis showed that FOXM1 expression was negatively correlated with overall survival in osteosarcoma patients.2.The results of GEO database showed that the expression of FOXM1 in drug-resistant cells was higher than that in sensitive cells.CCK-8 results showed that the IC₅₀ value of the drug-resistant cells was 4 times higher than that of the sensitive cells,indicating that the drug-resistant cells had drug resistance.Western blot results showed that the expression of FOXM1 in drug-resistant cells was significantly higher than that in sensitive cells.3.Western blot results showed that FOXM1 protein expression was significantly increased after MTX treatment in a concentration-and time-dependent manner.RT-q PCR results showed that the expression level of FOXM1 m RNA was gradually increased after MTX treatment.4.The results of Western blot and RT-q PCR showed that the expression levels of FOXM1protein and m RNA were significantly down-regulated and up-regulated in FOXM1knockdown and FOXM1 overexpression osteosarcoma cells.5.CCK-8 results showed that overexpression of FOXM1 enhanced the proliferation ability of osteosarcoma cells after MTX chemotherapy,while knockdown of FOXM1 reduced the proliferation ability of osteosarcoma cells after MTX chemotherapy.The results of flow cytometry showed that overexpression of FOXM1 reduced the apoptosis rate of osteosarcoma cells after MTX chemotherapy,while knockdown of FOXM1 increased the apoptosis rate of osteosarcoma cells after MTX chemotherapy.In addition,Western blot results showed that after MTX chemotherapy,overexpression of FOXM1 promoted the expression levels of cleave-caspase3 and Bax proteins in osteosarcoma cells,while knockdown of FOXM1 reduced the expression levels of cleave-caspase3 and Bax proteins in osteosarcoma cells.6.Western blot results showed that overexpression of FOXM1 promoted the level of autophagy,while knockdown of FOXM1 reduced the level of autophagy in osteosarcoma cells treated with MTX.In addition,immunofluorescence results showed that overexpression of FOXM1 increased the expression of LC3B fluorescent protein in osteosarcoma cells treated with MTX chemotherapy,while knockdown of FOXM1decreased the expression of LC3B fluorescent protein in osteosarcoma cells treated with MTX chemotherapy.7.The results of flow cytometry showed that inhibition of autophagy increased the apoptosis rate of FOXM1-overexpressing osteosarcoma cells after MTX chemotherapy.8.Co-immunoprecipitation showed that FOXM1 interacted with HMMR.Western blot results showed that FOXM1 protein positively regulated the expression of HMMR protein.9.The results of flow cytometry showed that overexpression of HMMR reversed the increase of apoptosis rate caused by FOXM1 knockdown in osteosarcoma cells treated with MTX.10.Western blot results showed that knockdown of FOXM1 decreased the expression levels of ATG7 and LC3-Ⅱprotein in osteosarcoma cells treated with MTX chemotherapy,while overexpression of HMMR reversed the expression levels of ATG7 and LC3-Ⅱprotein.11.Subcutaneous tumor formation experiment in nude mice showed that FOXM1knockdown mice had slower tumor growth rate,smaller tumor volume,and higher sensitivity to MTX than the control group.12.The results of TUNEL staining and immunofluorescence showed that the proportion of apoptotic cells in tumor tissues of FOXM1 knockdown mice was significantly higher than that of the control group after MTX chemotherapy,and the expression of LC3B green fluorescent protein was lower than that of the control group.Conclusion:FOXM1 induces MTX-resistant osteosarcoma cells by regulating HMMR-ATG7 axis to induce autophagy.Therefore,FOXM1 inhibitors may be an effective strategy to address the current chemoresistance in osteosarcoma for MTX-resistant patients.