| Glioma is the most common primary intracranial tumor with high mortality and morbidity.Glioblastoma(GBM)is the most malignant tumor,accounting for 47.7% of brain and other central nervous system malignant tumors,and 56.6% of gliomas.The treatments of glioma include postoperative radiotherapy and chemotherapy,which are prone to develop radioresistance and drug resistance,respectively.Studies have found that the stability of iron metabolism is of great significance for maintaining the normal function of cells,and is closely related to the occurrence and development of tumors.Iron metabolism in cells mainly includes the transfer,utilization and export of iron.Proteins involved in cellular iron transport and storage include transferrin receptor 1(TFR1),ferroportin(FPN),divalent metal transporter 1(divalent metal transporter 1),and transferrin receptor 1(TFR1).DMT1)and ferritin.Iron regulatory protein 1(iron regulatory protein 1,IRP1)and iron regulatory protein 2(iron regulatory protein 2,IRP2)regulates iron metabolism by binding to iron regulatory elements(IRE)on the m RNA of iron metabolism-related proteins mentioned above,thereby maintaining intracellular iron stability.In previous studies,we found that the iron contents in glioma tissues were reduced in comparison with normal brain tissues,but the mechanism is unknown.It is of great significance to explore the regulatory mechanism of iron metabolism in glioma for the development of treatment.To investigate the regulatory mechanisms of iron metabolism-related proteins in glioblastoma,human normal astrocytes HA1800 and human GBM U87 cells were used in this experiment.The iron content between HA1800 cells and U87 cells was first measured using an iron content assay kit.Western blotting was used to detect the expression levels of iron metabolism-related proteins in HA1800 and U87 cells.The m RNA expression levels of iron metabolism-related genes in HA1800 and U87 cells were detected by real-time fluorescence quantitative PCR.In order to determine whether iron treatment has an effect on iron metabolism and the function and behavior of U87 cells,U87 cells were treated with ferric ammonium citrate(FAC)at different concentrations.CCK-8 and trypan blue staining were used to observe the survival changes of U87 cells after FAC treatment.The expression levels of iron metabolism-related proteins in U87 cells treated with FAC were detected by western blotting.Cell scratch assay,Transwell migration and invasion assay were used to detect the migration and invasion of U87 cells after FAC treatment.Transmission electron microscopy was used to observe the mitochondrial morphology of cells.Flow cytometry,PI staining and western blotting were used to detect apoptotic proteins,which reflect the level of lipid peroxidation and apoptosis.The results were as follows:1.Compared with HA1800 cells,the iron content of U87 cells was reduced by 66%,and the difference was highly statistically significant(P<0.001).2.Compared with HA1800 cells,the m RNA expression levels of IRP1,FPN,DMT1 and FTH in U87 cells were increased by 91.4%,103.3%,27.8% and 93.1%,respectively,with statistically significant differences(P<0.05,P<0.01,P<0.001).The m RNA expression levels of IRP2 and TFR1 in U87 cells were decreased by 44.6% and 82%,respectively,with statistically significant difference(P<0.05,P<0.01).3.Compared with HA1800 cells,the protein expression of TFR1,FPN and DMT1 in U87 cells increased by 200.1%,655.4% and 203%,respectively,with statistically significant differences(P<0.05,P<0.01,P<0.001).The protein expression of IRP2,IRP1 and FTH in U87 cells decreased by 56.4%,65.3% and 49.3%,respectively,with statistically significant differences(P<0.05,P<0.01).4.U87 cells were treated with different concentrations of FAC(0.5 μM to 100 μM)for 24 hrs.CCK-8 results showed that,compared with the control group,The cell viability of 5μM,10 μM,20 μM,30 μM,40 μM,50 μM and 100 μM groups decreased by 38.5%,43%,60%,62.2%,67.7%,80% and 99.7%,respectively,and the differences were highly statistically significant(P<0.001).5.Compared with the Na Cl group,the protein expression of IRP2,IRP1,TFR1,FPN and DMT1 in U87 cells treated with 5 μM FAC for 24 hrs decreased by 32.4%,67.7%,36.9%,74.2% and 70.2%,respectively(P<0.05,P<0.01).However,the protein expression of FTH increased by 88.1%,and the difference was highly statistically significant(P<0.001).6.Compared with the Na Cl group,U87 cells treated with 5 μM FAC for 24 hrs showed decreased proliferation ability,and the proportion of trypan blue staining positive cells increased by 30%(P<0.001).7.The results of cell scratch test showed that compared with the Na Cl group,the migration rate of U87 cells treated with 5 μM FAC decreased by 16%,and the difference was highly statistically significant(P<0.01).The results of Transwell migration assay showed that compared with the Na Cl group,the migration rate of U87 cells treated with 5 μM FAC was decreased by 50.6%,and the difference was highly statistically significant(P<0.001).8.The invasive ability of U87 cells after FAC treatment was detected by Transwell invasion assay.Compared with the Na Cl group,the invasion rate of U87 cells treated with 5 μM FAC for 24 hrs decreased by 27.7%,and the difference was highly statistically significant(P<0.01).9.Compared with the Na Cl group,mitochondria damage was observed in U87 cells treated with 5 μM FAC for 24 hrs.10.Compared with the Na Cl group,the protein expression of GPX4 in U87 cells treated with5 μM FAC for 24 hrs decreased by 42.8%,and the difference was highly statistically significant(P<0.01).The lipid peroxidation level increased by 632.3%,and the difference was highly statistically significant(P<0.001).11.Compared with the Na Cl group,the Bcl-2/Bax protein ratio in U87 cells treated with 5μM FAC decreased by 34.3%,and the difference was statistically significant(P<0.05).The proportion of PI positive cells was increased by 32.4%,and the level of apoptosis was increased by 50.6%,the difference was highly statistically significant(P<0.001).In conclusion,compared with normal astrocytes,U87 cells have reduced iron content,increased expression of iron transporters,and decreased expression of iron regulatory proteins and iron storage proteins,this suggests that iron metabolism is altered.After FAC treatment,the iron metabolism of U87 cells was changed,the cell survival rate,cell proliferation,cell migration and cell invasion were decreased,the cell mitochondria were damaged,and the levels of lipid peroxidation and apoptosis were increased.This study helps to understand the important role of iron metabolism in the occurrence and development of GBM,and is of great significance for the development of new treatment methods for GBM. |
PDF Full Text Request