Preliminary Study On The Role Of Guanylate Binding Protein 1 Promoting Glioma Progression

【Objective】Guanylate binding protein 1(GBP1)is a large GTPase,which is induced by interferonγ(IFN-γ).It is widely expressed in all organs of the whole body and has been shown important for host immune defense against various pathogens.Recent studies have found that GBP1 is involved in the process of tumor development,invasion and metastasis,but the molecular mechanism is not clear,and the role in different tumors is also different.Our previous study found that GBP1 could promote the proliferation of human glioma cells in nude mice,but the proliferation of glioma cells did not change significantly in vitro.The aim of this study was to investigate the mechanism of GBP1 promoting glioma proliferation.【Methods】1.The relationship between the expression of GBP1 in glioma tissues and the survival of glioma patients was retrieved from the Rembrandt database.The relationship between GBP1 expression and CCL2 expression was also discussed.2.GBP1 was overexpressed in the glioma cell U87 with retroviral vector(U87-GBP1),the control cell was U87-LacZ.The expression of GBP1 in glioma cells LN229 was knowdown by lentivirus vector to establish the low expression cell LN229-shGBP1,and the control cell was LN229-shN.RNA and protein were extracted from the glioma cells.The mRNA and protein expression of GBP1 were analyzed by qRT-PCR and Western blot methods.The cell culture was harvested and stored at-70℃for further studies.3.The growth of glioma cells in vitro was analyzed by CCK-8 reagent.4.The expression of chemokine CCL2 in glioma cells and cell culture supernatants were detected by qRT-PCR and ELISA respectively.5.The human peripheral blood mononuclear cells(PBMCs)were separated using Ficoll-Paque and then to collecte the adherent cells which containing monocytes mainly.The monocytes were treated with glioma cell culture medium for 2 days,and the ratio of human myeloid derived suppressor cells(MDSCs)in monocytes was analyzed by flow cytometry.6.U87-GBP1 and LN229-shN cells were cultured in medium containing anti-human CCL2 antibody,ant then the glioma cell cultures were collected to culture the monocytes for 2 days.The proportion of MDSCs was analyzed by flow cytometry.7.5*10~5/10μL U87-GBP1 and U87-LacZ cells were inoculated in BalB/C nude mice with intracranial tumorigenesis.After 40 d,the mice were killed in euthanasia.The xenografts,bone marrows and spleen tissues of mice were separated,and the MDSCs ratio was analyzed by flow cytometry.【Results】1.The results of database showed that the expression of GBP1 in human glioma tissues was inversely correlated with the survival of glioma patients,and positively correlated with CCL2 expression.2.qRT-PCR and Western blot were used to analyze the mRNA and protein expression of GBP1 in the glioma cells expressing or low expression of GBP1.The results showed that the expression of GBP1 in U87-GBP1 cells was significantly higher than that of the control cell U87-LacZ,and the GBP1 expression in LN229-sh GBP1 was lower than that of the LN229-shN cells.The glioma cells with overexpression or silencing of GBP1 were successfully established.3.The proliferation of glioma cells were detected by CCK-8 method.Compared with the control cells,the proliferation of U87-GBP1 did not change significantly after overexpression of GBP1,and the proliferation of LN229-shGBP1 cells did not change significantly after knockdown GBP1.4.The mRNA expression of CCL2 in U87-GBP1 cells and the expression of CCL2 protein in cell culture supernatant increased respectively and the expression of CCL2 in LN229-shGBP1 cells and cell culture supernatant decreased after silencing GBP1.5.The proportion of MDSCs in normal monocytes treated with glioma cell culture medium was significantly changed,in which the U87-GBP1 group was higher than that of the control cell U87-LacZ,and the LN229-shGBP1 group was lower than the LN229-shN cell.6.U87-GBP1 and LN229-shN cells were cultured in medium containing anti-human CCL2 antibody,and the cultures were collected to treat monocytes.The ratio of MDSCs in monocytes was decreased.7.U87-GBP1 and U87-Lac Z cells were orthotopic implanted in nude mice.U87-GBP1 xenografts were significantly larger than that of U87-Lac Z.The proportion of MDSCs increased significantly in xenograft,bone marrow and spleen tissues of U87-GBP1 bearing mice than that of U87-LacZ bearing mice(P<0.05).【Conclusions】1.Overexpression of GBP1 or low expression of GBP1 did not affect the proliferation of human glioma cells U87 and LN229 in vitro.2.The expression of GBP1 in human glioma tissues was positively correlated with CCL2 expression.GBP1 might promote the expression of chemokine CCL2 in glioma cells.3.Glioma cells might induce normal monocytes to acquire MDSCs-like phenotype in vitro,and chemokine CCL2 induced by GBP1 may be involved in it.4.The experiment results in vivo suggested that GBP1 might induce the increase of MDSCs to develop an immunosuppressive microenvironment in the glioma microenvironment,thereby promoting glioma proliferation.