Studies On The Expression Of Gpx4 And Its Function In Glioma

ObjectiveGlioma is one of the most common and aggressive types of human brain tumors,and it is of great significance to explore novel glioma related genes.The purpose of this research is to study the expression of Gpx4 at the level of tissue in glioma patients,and analysis of Gpx4 and clinical pathological features of patients with glioma and the correlation between the expression of proliferation markers Ki-67,and to investigate the role of Gpx4 in the development and progression of glioma.In addition,the effects of Gpx4 on proliferation,migration and apoptosis of glioma cells were investigated at the cytological level,and explore the possible mechanism of Gpx4 regulating glioma cell growth.Methods1.The expression of Gpx4 in normal brain tissues and glioma tissues was detected by Western blot?Western,blot,WB?.Immunohistochemistry?IHC?was used to explore the expression of Gpx4 and Ki-67 on 81 paraffin-embedded slices.The associations between Gpx4 expression and clinicopathologic characteristics were analyzed by Pearson?² test.The Kaplan-Meier survival curve was used to analyze the effect of Gpx4 expression on the prognosis of patients with glioma.2.In different glioma cell lines?A172,H4,U87,U251 and U118?,the expression of Gpx4 was detected by WB,and the cell lines with high expression of Gpx4 were screened out.Gpx4 glioma cell line was selected,and the small interfering plasmid was transiently transfected into U87,and the highest interference efficiency plasmid was screened out.3.Gpx4-siRNA#1 was transfected into U87 glioma cells,and WB was performed to detect the effect on PCNA and cyclinD1.Flow cytometry was performed to analyze the influence on cell cycle distribution.Cell vitality was determined by Cell Counting Kit?CCK8?assay,and colony formation analyses was employed to explore the effect on cell proliferation.4.Wound-healing and transwell assays were used to investigate cell migration.Meanwhile,the expression of some proteins related to migration was analyzed by WB.5.The apoptosis of the cells in the control group and the small interfering group was detected by flow cytometry,in order to analyze the effect of interfering Gpx4 on the apoptosis of U87 cells.Meanwhile,the expression of apoptosis related proteins was detected by WB,so as to facilitate the further study of possible molecular mechanisms.Results1.Western blot showed that the expression of Gpx4 in glioma tissues was significantly higher than that in normal brain tissues,and the expression of Gpx4 was gradually increased with the increase of WHO grade in gliomas.The results of immunohistochemistry showed that the expression of Gpx4 was significantly correlated with Ki-67 and WHO classification,but had no obvious correlation with other pathological features.The protein expression levels of Gpx4 were different in different grades of glioma tissues.The Kaplan-Meier survival curve analysis showed that the overall survival rate of patients with higher Gpx4 expression was significantly lower than that in patients with low Gpx4 expression?P<0.05?.2.In A172,H4,U87,U251,and U118 glioma cells,Gpx4 was found to be the highest expression in U87 cells by WB.After transfection of small interfering plasmids of Gpx4into U87 cells,the interference of Gpx4-siRNA#1 was found to be the most efficient.3.WB analysis found that after knockdown Gpx4 protein expression,cell proliferation index of PCNA and cyclinD1 were reduced,and flow cytometry analysis showed that knockdown of Gpx4 would increase the cells of G1 phase,while the number of cells in S phase decreased.Cell Counting Kit?CCK8?assay and colony formation analyses showed that the interference of Gpx4 expression could inhibit the proliferation of glioma cells.4.Wound-healing and transwell assays showed that knockdown of Gpx4 can inhibit the cell growth,and WB results showed that inhibition of the expression of Gpx4 will promote the expression of E-cadherin,while the expression of Vimentin was inhibited.5.The results of flow cytometry showed that the number of apoptotic glioma cells increased after interfering the expression of Gpx4.The results of WB showed that knockdown of Gpx4 could promote the expression of cleaved caspase 3,accompanied by decreased expression of Bcl-2.Conclusions1.Gpx4 was overexpressed in glioma tissues,and the expression partten of Gpx4 was positively related to Ki-67 expression and glioma pathological grade.The expression level of Gpx4 is closely associated with the prognosis of patients,and high expression of Gpx4may result in poor prognosis of glioma patients.2.Gpx4 can modulate the process of glioma cell proliferation,migration and apoptosis,and knockdown of Gpx4 expression would lead to cell cycle arrest,and reduce the cell migration,and also induce apoptosis.