PLA2G16 Promotes Osteosarcoma Metastasis And Drug Resistance Via MAPK Pathway

ObjectiveClarify the correlation between the expression of PLA2G16 gene and the development and metastasis of human osteosarcoma.Identify the underlying mechanism of PLA2G16 in regulating osteosarcoma progression and metastasis to determine the value of PLA2G16 in osteosarcoma therapy,and establish PLA2G16 as a clinic biomarker for osteosarcoma prognosis.Methods1)Use of lentivirus or retroviral packaging system to establish PLA2G16 stable overexpressed or knockdown osteosarcoma cell lines to analyze the effect of PLA2G16 expression on osteosarcoma cell proliferation(MTT assay),migration(wound-healing assay),invasion(Matrigel Transwell assay),Colonigenicity(Colony Formation assay),anchorage-independent growth(Agar soft assay)and drug sensitivity.Meanwhile,get the mutant PLA2G16-C113 S gene which lost the phosphorylapase activity of PLA2G16 to identify its influence on the phenotype change of osteosarcoma cells.2)Explore the potential mechanism of PLA2G16 in promoting osteosarcoma development and metastasis by real time PCR and western blot or other techniques.Then inhibit the expression of PLA2G16 and its target proteins by small molecule inhibitors to further confirmed that PLA2G16 can promote the development and metastasis of osteosarcoma because of its phospholipase activity and the effect of the target proteins.3)Subcutaneously injected PLA2G16 stably overexpressed cell lines into nude mice to observe the effect of PLA2G16 on tumonigenicity in vivo,and detect the expression of PLA2G16 and its downstream protein by western blot analysis.4)Determine the expression of PLA2G16 in osteosarcoma tissue by immunohistochemistry and tissue microarray technology,and analyze the correlation between the expression of PLA2G16 and its downstream regulatory proteins in the progression and metastasis of osteosarcoma.Results1)PLA2G16 promotes osteosarcoma cell proliferation,colony formation,migration and invasion because of its phosphorylipase activity;2)The expression of PLA2G16 decreased the sensitivity of osteosarcoma cells to chemotherapeutic drugs in time-and dose-dependent manner;3)The oncogenic function of PLA2G16 is associated with cytoplasmic localization and is partially mediated through activation of MAPK signaling in osteosarcoma cells;4)PLA2G16 contributes tumorigenesis in vivo;5)PLA2G16 expression is associated with ERK1/2 phosphorylation in osteosarcoma patients and predicts for poor overall and metastatsis-free survival.Conclusion1)PLA2G16 acts as an oncogene in promoting the progression and metastasis of osteosarcoma by activating MAPK pathway.2)PLA2G16 can be used as a biomarker for the diagnosis and treatment of osteosarcoma.