| BackgroundGlioblastoma(GBM),as one of the most common malignant primary tumors in central nervous system,is associated with high mortality and recurrence rate owing to the aggressive nature.Despite of the active standard treatment including maximal resection followed by radiotherapy with concomitant chemotherapy and adjuvant targeted therapy and immunotherapy,the prognosis is still poor.MAGED4,as a member of melanoma associated antigen(MAGE)family,is highly expressed in a variety of tumor tissues including glioma,but it is rarely detected in a variety of normal tissues,so it is expected to become an ideal candidate target for tumor therapy,but the function and mechanism of MAGED4 in GBM are still unknown.ObjectiveTo explore the function of MAGED4 in GBM,identify the regulatory mechanism of MAGED4 on its potential downstream molecules,and analyze their clinical significance in glioblastoma patients.Methods1.The potential biological functions and the downstream targets of MAGED4 in glioblastoma were analyzed by functional analysis of co-expressed genes of MAGED4 in GBM samples of Linked Omics database and differentially expressed genes of RNA-seq in the glioblastoma cell line U87-MG with downregulated MAGED4.2.The expression levels of the downstream molecules were detected by qPCR and WB.The biological function of MAGED4 in GBM was verified by plate colony formation assay,CCK-8 assay,cell cycle distribution detection by flow cytometry,WB detection of cell cycle related proteins,establishment of transplanted tumor model in nude mouse and immunohistochemical staining(IHC)of the transplanted tumor tissue sections.3.QPCR and WB were used to detect the expression of MAGED4 and CDC25A after bioinformatics prediction of shared target miRNA of both MAGED4 and CDC25A and transfection of mimics and inhibitors of the target miRNA in GBM cell lines.Fluorescence in situ hybridization,dual luciferase reporter assay and RNA immunoprecipitation(RIP)were performed to explore the effect and regulation mechanism of MAGED4 on CDC25A in GBM;4.QPCR was used to detect the expression of MAGED4 and CDC25A mRNA in glioblastoma specimens.Prognosis analysis and correlation analysis between MAGED4 and CDC25A expression with clinicopathological features were performed to analyze the clinical significance of MAGED4 and CDC25A in glioblastoma patients and samples from TCGA and CGGA databases.Results1.According to the differential analysis of expression profile fromRNA-seq in GBM cell line U87-MG with downregulated MAGED4,219 up-regulated genes and 171 down-regulated genes were identified.The functional analysis results of the co-expressed genes of MAGED4 in GBM samples from Linked Omics database and top 100 differentially expressed genes of RNA-seq suggested that MAGED4 may be involved in the regulation of glioblastoma cell proliferation and cell cycle.According to the results of functional enrichment and correlation analysis of MAGED4 expression in glioblastoma samples from GEPIA2 and CGGA databases,CDC25A was considered as the downstream target of MAGED4 in GBM.2.The detection of CDC25A showed that the CDC25A expressions were decreased in GBM cell lines U87-MG and U251 with the down-regulation of MAGED4.Results of experiments both in vivo and in vitro showed that interference of MAGED4 in GBM could inhibit the proliferation and tumorigenesis of GBM both in vivo and in vitro and MAGED4 could regulate the cell cycle of GBM cells through CDC25A.3.The prediction results of Target Scan database showed that there were specific binding sites of miR-200a-3p on the 3 ’-UTR of both MAGED4 and CDC25A mRNA.Fluorescence in situ hybridization results showed that miR-200a-3p was co-located with the mRNA of MAGED4 and CDC25A in the cytoplasm of GBM cell line U87-MG,respectively.The expression levels of MAGED4 and CDC25A were decreased in U87-MG after transfection of miR-200a-3p mimics and were increased in A172 after transfection of miR-200a-3p inhibitor.The results of dual luciferase reporter assay and RIP showed that miR-200a-3p inhibited the expression of MAGED4 and CDC25A by targeting the3’UTR of MAGED4 and CDC25A mRNA in GBM.The CDC25A mRNA expression was decreased after transfection of siRNA-MAGED4 and was reincreased after co-transfection of siRNA-MAGED4 and miR-200a-3p inhibitor both in U87-MG and U251.Consistently,the MAGED4 mRNA expression was decreased after transfection of siRNA-CDC25A in U87-MG,and was reincreased after co-transfection of siRNA-CDC25A and miR-200a-3p inhibitor in U87-MG.But there was no significant change of MAGED4 mRNA expression in U251 after transfection of siRNA-CDC25A.It was suggested that the regulation of MAGED4 on CDC25A depended on miR-200a-3p in GBM,and MAGED4 and CDC25A may be ceRNAs and regulate the expression of each other by competing for miR-200a-3p.4.The mRNA levels of MAGED4 and CDC25A were both up-regulated in GBM specimens.Patients with high MAGED4 level had a poor prognosis compared to those with low MAGED4 level,furthermore,cox regression analysis revealed that high expression of MAGED4 and non-acceptance of radiotherapy or chemotherapy were independent poor prognosis factors for GBM patients.In contrast,there was no prognostic significance of CDC25A in GBM patients.The correlation analysis showed that the MAGED4 mRNA level was associated with recurrence in GBM patients with IDH-wildtype in mRNAseq＿693 dataset of CGGA database and was associated with P53 protein level in GBM patients.ConclusionThis study confirmed that MAGED4 regulates the expression of CDC25A through competitive binding with miR-200a-3p and affects the cell proliferation and cell cycle in glioblastoma.MAGED4 and CDC25A are highly expressed in glioblastoma tissues.The expression of MAGED4 was associated with the clinicopathological parameters in GBM patients and was an independent prognostic factor for GBM patients.In consideration of the function and expression specificity of MAGED4 in glioblastoma,it may be a potential prognostic and therapeutic target for glioblastoma. |
