The Effect And Clinical Significance Of FNDC4 On The Biological Behavior Of Glioblsatoma And The Polarization Of Macrophages

Objective:Glioblastoma is a common and highly malignant tumor in the central nervous system.At present,the clinical treatment of patients with glioblastoma mainly includes surgical resection combined with postoperative radiotherapy and adjuvant combined therapy with temozolomide and other drugs.Although with the continuous in-depth research and exploration of glioma by researchers,the overall research progress and the actual effect of clinical treatment are still worrying.According to literature,the 5-year survival rate of glioblastoma patients is less than 10%.Therefore,from the perspective of molecular level,it is urgent to find effective therapeutic targets to improve the therapeutic effect of patients.Fibronectin Type III Domain Containing protein(FNDCs)is a highly conserved protein in structure,which is involved in many physiological and pathological processes such as cell metabolism,inflammation,cardiovascular diseases and tumors in human body.FNDC4 is an important member of fibronectin type III domain protein family and is highly expressed in brain tissue.Studies have shown that FNDC4 can specifically bind macrophages to affect the function of macrophages and play an anti-inflammatory role.Currently,there are no studies on FNDC4 in glioblastoma.,therefore,the aim of this study was to explore FNDC4 expression differences in the patients with neurological glioblastoma level and its effect on the prognosis of patients,the neurological glioblastoma cell biology and behavior of macrophage,and the influence of the polarization effect of the three levels to explore FNDC4 neural glioblastoma prognostic effects and its mechanism.Method:1.Download the m RNA expression data and survival data of glioblastoma tumor tissue from TCGA,GTEx and CGGA databases,and use R language to perform maximum selection rank statistical analysis and survival analysis on the expression of FNDC4 and patient survival data.2.Using lentivirus to construct FNDC4-overexpressing glioblastoma cell lines U-87 MG and U251,and the expression and secretion of FNDC4 were detected by Western-blot and ELISA.3.Using CCK-8 kit to detect the effect of FNDC4 overexpression on the proliferation activity of glioblastoma cell lines U-87 MG,U251 and the sensitivity to chemotherapeutic drug BCNU.4.Using Annexin V-PI apoptosis kit and flow cytometry to detect the effect of FNDC4overexpression on the apoptosis of glioblastoma cell lines U-87 MG and U251.5.Using cell cycle kit and flow cytometry to detect the effect of FNDC4 overexpression on the cell cycle of glioblastoma cell lines U-87 MG with or without BCNU.6.Collect peripheral blood from healthy volunteers,and separate peripheral blood mononuclear cells(PBMCs)by gradient centrifugation,further sort CD14~+cells from peripheral blood mononuclear cells.7.CD14~+PBMCs were cultured with macrophage colony-stimulating factor(M-CSF)to induce their differentiation into M0 macrophages.8.Using LPS and IL-4 to stimulate M0 macrophages and induce their polarization to M1and M2 macrophages,respectively.9.Add exogenous FNDC4 to M0,M1,M2 polarized macrophages respectively,and use flow cytometry to detect the effect of exogenous FNDC4 on macrophage polarization.10.The FNDC4-overexpressing glioblastoma cell lines U-87 MG and U251 were co-cultured with M1-polarized macrophages,and their effects on M1-polarized macrophages were detected by flow cytometry.Result:1.TCGA database was combined with GTEx database for differential expression analysis,and the results showed that FNDC4 was significantly differentially expressed in glioblastoma and normal brain tissues(p=1.67e-13).Survival analysis showed that the expression of FNDC4 was negatively correlated with the prognosis of patients with glioblastoma,and there was statistical significance(p=0.01).The difference analysis of CGGA database indicated that FNDC4 was significantly differentially expressed in glioblastoma and normal brain tissues(p=4.71e-10).The results of survival analysis indicated that the prognosis of patients with low FNDC4 expression was better than that of patients with high FNDC4 expression,and the difference was statistically significant(p=0.015).2.The glioblastoma cells U-87 MG and U251 overexpressing FNDC4 were successfully constructed.Western-blot and ELISA results showed that the expression and secretion of FNDC4 in U-87 MG and U251 cells overexpressing FNDC4 were significantly increased.3.The results of CCK-8 experiment indicated that FNDC4 overexpression had no significant effect on the proliferation activity of glioblastoma cell lines U-87 MG and U251,and did not affect their sensitivity to BCNU.4.Flow cytometry results of apoptosis showed that FNDC4 overexpression had no significant effect on the apoptosis of glioblastoma cell lines U-87 MG,U251 with or without BCNU.5.The results of flow cytometry analysis showed that FNDC4 overexpression had no significant effect on the cell cycle of glioblastoma cell line U-87 MG with or without BCNU.6.Flow cytometry results showed that exogenous FNDC4 inhibited the polarization of M1macrophages,but had no significant effect on the polarization of M2 macrophages.7.The results of flow cytometry showed that co-culture of FNDC4-overexpressing glioblastoma cell lines U-87 MG and U251 with M1 macrophages could significantly inhibit the polarization of M1 macrophages.Conclusion:The expression of FNDC4 in glioblastoma tumor tissue is negatively correlated with the prognosis of patients;overexpression of FNDC4 had no effects on the proliferation,apoptosis,cell cycle and the sensitivity to BCNU of glioblastoma cells;the reason that FNDC4 affects the prognosis of glioblastoma patients could possibly be that FNDC4 inhibit the polarization of M1 macrophages.