| Objective: Among primary intracranial tumors,gliomas are by far the most common.The World Health Organization grades gliomas into I to IV.Grade I and II are called low-grade gliomas,and grade III and IV are called high-grade gliomas.According to literature reports,the 50% survival time of patients with grade IV glioma after surgery combined with radiotherapy and chemotherapy is still only about 430 days.How to solve the difficult problem of glioma treatment has become the primary task of scientists and clinicians.The Forkhead box(FOX)protein family is a protein factor involved in the transcription process,which mainly plays a role in the DNA binding region of eukaryotes.The abnormal expression of FOXA1 gene is related to the progress of many diseases,such as breast cancer and prostate cancer.However,its role in glioma remains to be further explored.Therefore,exploring and elucidating the specific mechanism of the FOX family in the development of glioma is of great significance for improving the prognosis of glioma.Methods: 1.Construction of prognostic risk signature and screening of core member genes a)We download and process two public databases containing glioma patient information,including The Cancer Genome Atlas(TCGA),Chinese Glioma Genome Atlas(CGGA).b)In TCGA and CGGA,according to the previous literature reports,all FOX family genes were sorted out,and an IDH1 wild-type glioblastoma prognostic risk signature was constructed through LASSO analysis,and the prediction effect was verified.Then,the relationship between each member in this signature and the clinical and pathological characteristics of glioma patients was analyzed through bioinformatics methods such as heat map and correlation analysis.c)Through correlation analysis and protein interaction analysis tool STRING,we analyzed the relationship between the member genes of the FOX family prognostic risk signature.And we screen out the core member gene is FOXA1 through uni-COX analysis in the TCGA RNA-seq database and CGGA microarray database.d)In the TCGA RNA-seq database and the CGGA microarray database,the relationship between FOXA1 and the survival time of glioma patients was analyzed.2.Detection of the expression of the core member gene FOXA1 of the prognostic risk signature and its impact on phenotypic function a)Normal human astrocytes,U87 cells and U251 cells were cultured in vitro.The expression of FOXA1 in normal human astrocytes and glioma cells was detected by q PCR and Western Blot.b)The FOXA1 gene in the tumor cells was knocked down by lentiviral transduction.The knockdown efficiency of FOXA1 was detected by q PCR and Western Blot.c)After the FOXA1 knockdown in the tumor cells is completed,the phenotypic changes of the tumor cells were detected.3.Determination of the function of the FOXA1 a)In the TCGA RNA-seq database and the CGGA microarray database,the correlation analysis and difference analysis of FOXA1 were carried out,and the intersection of the two was taken to obtain the intersection gene.b)Annotate these genes through Gene Set Enrichment Analysis(GSEA)and Kyoto Encyclopedia of Genes and Genomes(KEGG)to determine the main function of the intersection genes,which is also the function of the FOXA1.4.The downstream target gene of FOXA1 is XRCC2 a)After clarifying the function of FOXA1,in order to find the downstream genes of FOXA1 more accurately,we selected the top 10 genes with the highest correlation with FOXA1 in TCGA RNAseq and CGGA microarray respectively,and displayed them in the form of a heat map.b)In TCGA RNA-seq and CGGA microarray,according to the median expression value of FOXA1,patients data was divided into high expression group and low expression group,and the top 10 genes with significant differences between groups(10genes with the smallest P value)were selected and presented in the form of a heat map.c)We intersected these results above again,and selected the most likely downstream gene of FOXA1 from them.V.Determination of the relationship between FOXA1 and its downstream target gene XRCC2 a)We analysed the correlation between FOXA1 and XRCC2(TCGA RNA-seq,CGGA microarray).b)At the same time,we verified whether the XRCC2 gene can be regulated by FOXA1 in three databases(JASPAR,Animal TFDB3,Cistrome DB(http://dbtoolkit.cistrome.org).6.Gene set enrichment analysis was to clarify the function of XRCC2.7.We analysed the relationship between XRCC2 and the clinical information and pathological characteristics of glioma patients.The analysis of XRCC2 expression and survival in different grades of glioma,and the survival analysis of patients who received radiotherapy and chemotherapy were completed.Results: 1.In TCGA RNA-seq and CGGA microarray,a prognostic risk signature of IDH1 wild-type glioblastoma was constructed,and it was found that it was correlated with the survival time,chrome 1p 19 q non-codeletion,high grade of glioma patients.And the core member gene of this signature is FOXA1.The analysis found that FOXA1 was associated with poor prognosis in glioma patients.2.Compared with normal human astrocytes,there is a higher expression of FOXA1 in glioma cells such as U87 cells and U251 cells.3.FOXA1 is related to some phenotypic functions of glioma cells,such as migration ability,invasion ability and proliferation ability.It can also participate in biological processes such as "mitosis,cell cycle" of cells.4.Through correlation analysis,it is found that the downstream target gene of FOXA1 is XRCC2,and FOXA1 can promote the expression of XRCC2 by combining with the promoter sequence of XRCC2.5.The functions of XRCC2 mainly focus on DNA replication and DNA damage repair.High expression of XRCC2 was positively associated with poor survival status,high grade,IDH1 wild-type status,and chrome 1p 19 q co-deletion in glioma.XRCC2 has a higher expression in glioma cells,and it is related to poor prognosis and resistance to radiotherapy and chemotherapy in glioma patients.Conclusion: We analyzed the FOX family members in TCGA RNA-seq IDH1 wildtype GBM according to the prognosis-related information by Lasso regression analysis,and constructed a risk signature related to prognosis,which can effectively predict the prognosis of glioma patients.The core member gene of this risk signature is FOXA1,which is clinically related to the poor prognosis of glioma patients and can positively promote the phenotypic functions of glioma cells,such as migration ability,invasion ability,and proliferation ability.The downstream target gene of FOXA1 is XRCC2,and the FOXA1 can promote the expression of XRCC2 at the m RNA level and protein level by binding to the promoter sequence of XRCC2.XRCC2 has a higher expression in glioma cells,and there is a positive relationship between it and the clinicopathological characteristics of glioma patients,and XRCC2 is related to the poor prognosis,radiotherapy resistance and chemotherapy resistance of glioma patients. |
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