Study On The Role And Molecular Mechanism Of MOXD1 Gene In The Proliferation Of Osteosarcoma

Objective:To study the role of MOXD1 in the occurrence and development of osteosarcoma based on the in vivo and vitro experiments.In addition,to explore the new idea and possible targets of biological treatment of osteosarcoma.Methods:1.Immunohistochemistry was used to detect the expression of MOXD1 in human osteosarcoma wax block tissue specimen.2.Three commonly used osteosarcoma cell lines were detected by RT-PCR.According to the expression abundance of MOXD1 in the cell lines,the two cells with highest expression abundance(U2OS and MNNGHOS Cl #5)were selected as experimental cells.3.Using the gene sequence of MOXD1 as template,RNA interference target sequence was designed to construct target gene RNA interference lentivirus vector.4.Osteosarcoma cells were transfected with RNAi lentivirus vector to construct a MODX1 lower-expression model.5.The knockdown efficiency of MOXD1 was detected by qPCR.Western blot was used to detected the expression of endogenous protein of MOXD1 and to determine the effectiveness of knockdown.6.Cell group:(1)shCtrl group: normal target cells plus negative control virus infected cells.(2)shMOXD1 group: normal target cells with MOXD1 gene shRNA virus infection.7.Cell proliferation of osteosarcoma cells in shCtrl and shMOXD1 group was detected by celigo cell count and MTT.8.The apoptosis of osteosarcoma cells in shCtrl and shMOXD1 group was detected by FACS apoptosis assay.9.The clonogenesis ability of osteosarcoma cells in shCtrl group and shMOXD1 group was detected by crystal violet method.10.Twenty nude mice were randomly divided into two groups,10 in each group.MNNGHOS Cl #5 osteosarcoma cell suspension of shCtrl group and shMOXD1 group were subcutaneously injected into axillary region of nude mice for in vivo osteosarcoma model construction,and the difference in growth status of transplanted tumor before and after MOXD1 knockdown was detected11?MNNGHOS Cl #5 cells from shCtrl group and shMOXD1 group were selected to extract Total RNA for quality control.The differentially expressed genes were detected by gene chip.q-PCR and Western blot detecte the expression differences of related genes.The differentially expressed genes were bioinformatics analyzed and the cell pathways related to MOXD1 were explored.Results:1?Immunohistochemical examination showed that the expression of MOXD1 in the tumor tissues was significantly increased in the samples of human osteosarcoma,and the expression was decreased or even not expressed in the adjacent tumor tissues and normal bone tissues.2?After transfection of osteosarcoma cells with RNAi lentivirus vector,MOXD1 gene in two osteosarcoma cells in shMOXD1 group was effectively knocked down compared with shCtrl group.3?Compared with the shCtrl group,the proliferation efficiency of MOXD1 in two osteosarcoma cells in the shMOXD1 group significantly decreased,and the number of apoptotic tumor cells significantly increased,and the number of clonal formation of osteosarcoma cells decreased(P<0.05).4?In the tumorigenosis experiment in nude mice,compared with shCtrl group,the number and tumor weight of MNNGHOS Cl #5 osteosarcoma cells in shMOXD1 group were significantly reduced,and the fluorescence expression of fluorescence imaging in living animals was decreased(P<0.05).5?Microarray analysis showed that 285 genes were up-regulated and 331 genes were down-regulated in the shMOXD1 group.MOXD1 regulates cellular pathways including EIF2,ERK5,and RAN,with the most significant inhibition of EIF2 cellular pathways.6?q-PCR and Western blot analysis of 37 genes related to proliferation and apoptosis showed decreased expression of PDGFRA,FOXM1,CCND3 and TNFAIP2 and increased expression of EGR1 in shMOXD1 group.The expression of CCND3 and TNFAIP2 was significantly decreased,while the expression of EGR1 was significantly increased.7?Western blot analysis was performed on the proliferation-related genes PCNA and cyclinD1,as well as the genes associated with the EIF2 pathway(p-EIF2?,EIF2?,EIF4 E,DDIT3 and AKT1),and the low expression of these genes was found.Conclusion:1 ? The low expression MOXD1 cell model was successfully conducted by transfection of osteosarcoma cells with RNAi lentivirus vector.2?The expression of MOXD1 in the paraffin specimens of human osteosarcoma showed obvious tumor aggregation,and there was no correlation between the expression of MOXD1 and the patient's gender,age,tumor site.The positive expression rate of MOXD1 was positively correlated with tumor size and Ennecking stage,and MOXD1 played an important role in the occurrence and development of osteosarcoma.3?An animal model of osteosarcoma was successfully constructed using the osteosarcoma cell line MNNGHOS Cl #5.Inhibition of gene MOXD1 can effectively inhibit the growth of osteosarcoma xenograft tumor.4?Knockdown MOXD1 can significantly inhibit the EIF2 cell pathway,leading to increased apoptosis of osteosarcoma cells,thus played an important role in the occurrence and development of osteosarcoma5?The gene MOXD1 may play a important role in osteosarcoma by regulating the expression of CCND3?TNFAIP2 and EGR2.6 ? It is the first time found that MOXD1 plays the role of oncogene in osteosarcoma,and it may be a potential target for the treatment of osteosarcoma.