| Glioblastoma(GBM)is the most malignant glioma,accounting for more than 50% of the incidence of glioma.Due to the high heterogeneity of GBM tumor cells,which are often characterized by diffuse infiltrating growth,and the serious vascularization in the tumor,it is difficult to achieve complete tumor resection in surgery,and GBM has a high tolerance to existing radiotherapy and chemotherapy,and a variety of combined treatment schemes have a poor effect on GBM.Therefore,it is of high research value to explore new treatment methods for GBM.Iron is an important trace substance for cell survival and metabolism,and participates in biological processes such as DNA synthesis,erythropoiesis,mitochondrial biosynthesis,energy metabolism and oxygen transport.Studies have shown that iron content and iron metabolism related proteins in breast cancer,ovarian cancer and other tumor tissues change.For example,the expression of FPN(Ferroportin)is reduced in breast cancer,resulting in increased levels of unstable iron pool and enhanced tumor growth.Similarly,FPN was found to be decreased in prostate cancer and ovarian cancer.The excessive deposition of iron in cells causes the abnormal metabolism of cell lipid oxides catalyzed by iron ions or iron-containing enzymes to increase.In this process,a variety of inducers destroy the redox balance of cells,and a large number of lipid peroxidation products accumulate,resulting in cell iron death.p53 plays an important inhibitory factor role in many tumors.It can induce growth arrest or apoptosis determined by cell damage or physiological environment,including damaged cell apoptosis,maintaining genomic stability,inhibiting angiogenesis,and regulating cell metabolism and tumor microenvironment.Research shows that p53 regulates iron homeostasis by regulating several iron metabolism-related proteins,and some iron regulators also form a feedback regulation circuit with p53.At the same time,p53 inhibits the expression of GPX4 and enhances the sensitivity of cells to iron death by inhibiting the transcription of SLC7A11,inhibiting the cystine/glutamate transport system(system xc-).Therefore,the interaction between p53 and iron metabolism related protein and iron death may reveal the mechanism of iron metabolism change in cancer.To sum up,studying the effect of p53 on iron metabolism in GBM can provide new research ideas for the pathogenesis of GBM and may play an important role in the treatment of GBM in the future.However,the role of p53 in iron metabolism and iron death in GBM is still unclear.To explore the effect of p53 on iron metabolism and iron death in GBM and its related mechanism.Two GBM cell lines U87(p53 wild type)and U251(p53 mutant type)were selected for cell culture.The iron level in the two cells was determined by the cell iron staining of Iron Assay Kit;The iron metabolism related protein DMT1(Differential metal transporter 1),Tf R1(Transferrin receiver 1),IRP1(Iron Regulatory Protein 1),IRP2(Iron Regulatory Protein 2),FTH(Ferritin heavy chain 1),FPN and iron death related protein index SLC7A11(also known as x CT)between the two cells were verified by extraction of cell protein and Western Blotting analysis Whether there are differences in GPX4(Glutathione peroxidase 4);Real time fluorescence quantitative PCR was used to quantitatively analyze and compare the m RNA expression level of iron metabolism-related genes in the two cells;Cell survival rate was detected by CCK-8(Cell Counting Kit-8)kit;The changes and differences of protein expression between cells after Erastin treatment;The level of lipid peroxidation of cells treated with Erastin was detected by flow cytometry;CRISPER-Cas9 was used to knockout the TP53 gene in U87 cells for detection of iron metabolism and ferroptosis related proteins.From the above results,we can get the difference of iron metabolism and iron death index between the two strains of cells with different p53 status,and judge the role of p53 in iron metabolism and iron death of GBM cell line based on the difference of the two strains of cells with different p53 types.The experimental results are as follows:1.Western blotting results showed that compared with U87 cells,the expression level of p53 protein in U251 cells decreased by 55.5%,with a statistically significant difference(P<0.05).2.Iron Assay Kit was used to determine the iron content of U87 and U251 cells.The results showed that the iron content of U251 cells was 21.6% higher than that of U87 cells,and the difference was statistically significant(P<0.05).3.RT-PCR results showed that compared with U87 cells,the m RNA expression levels of IRP1,IRP2,FPN,DMT1,and Tf R1 in U251 cells decreased by 46.49%,58.29%,92.88%,29.16% and 23.04%,respectively,with statistically significant differences(P<0.01).However,there was no significant difference in m RNA expression of FTH between the two types of cells(P>0.05).4.Western blotting results showed that compared with U87 cells,the FTH protein content in U251 cells decreased by 59.49%,the Tf R1 protein content increased by 23.17%,and the IRP2 protein content decreased by 40.80%,with statistical significance(P<0.05).However,there was no significant difference in the IRP1,DMT1,and FPN protein content(P>0.05).5.After 24 hours of treatment with Erastin,the survival rate of U251 cells decreased by 16.80% compared to U87 cells,with a statistically significant difference(P<0.05).The cell survival rates of 24 hours,48 hours,72 hours,96 hours,and 120 hours treated with Erastin were tested.The results showed that compared with the control group,the U87 group had a 10.72%,37.67%,49.64%,57.63%,and 55.55% decrease in cell survival rates,while the U251 group had a 10.33%,29.91%,54.36%,68.92%,and 86.38% decrease in cell survival rates;Compared with the DMSO group,the survival rate of the U87 group decreased by 11.40%,34.93%,48.72%,56.69%,and 56.07%,while the survival rate of the U251 group decreased by 8.97%,29.17%,54.55%,70.02%,and 86.11%,with statistical significance(P<0.05).6.After 24 hours of treatment with Erastin,the GPX4 protein expression in U87 cells decreased by 35.73% and 37.09% compared to the control group and DMSO group,respectively.The GPX4 protein expression in U251 cells decreased by 40.04% and 45.06%,respectively,and the differences were statistically significant(P<0.05).7.BODIPY C11 staining flow cytometry was used to measure the level of lipid peroxidation in cells.The results showed that after 24 hours of treatment with Erastatin,the lipid peroxidation level in U87 cells increased by 34.12% and 25.40% compared to the control group and DMSO group,respectively,with a statistically significant difference(P<0.01).The increase in U251 cells was 34.01%,respectively.And 31.38%,the difference was statistically significant(P<0.001).8.The results of transmission electron microscopy showed that in the U87 and U251 groups of cells,compared with the control group and DMSO group,the Erastin treatment group showed significant changes in cell morphology,with a significant increase in mitochondrial vacuoles,an increase in mitochondrial membrane density,a decrease in mitochondrial ridge structure,and nuclear wrinkling.9.After knocking out TP53 in U87 cells using CRISPER-Cas9 technology,Western blotting was used to determine iron metabolism and expression of iron death related proteins.The results showed that compared with the negative control group U87-NC cells,the expression levels of GPX4 and FTH in U87-TP53 KO cells decreased by 39.64% and 21.01%,respectively,after knocking out,and the differences were statistically significant(P<0.05).To sum up,different states of p53 can have different effects on the expression of iron metabolism-related genes and proteins in glioblastoma U87 and U251 cells,and the expression of iron metabolism-related proteins may have post-transcriptional modification and other processes.P53 mutation can cause the sensitivity of glioblastoma cells to iron death to increase,and the effect of this increased sensitivity may be caused by the inhibition of GPX4 expression.This study is of great significance to clarify the role of p53 in iron metabolism and iron death in glioblastoma,and provides a new idea for targeted treatment of glioblastoma. |
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