Transcriptional Regulation Of MAGE-D4 Mediated By P53 In Glioma

Objective To investigate the transcriptional regulation of p53 on glioma related gene MAGE-D4.Methods 1.Bioinformatics analysis:MatInspector online software was used to analyze the transcription factor p53 binding sites in the core promoter region of MAGE-D4.2.Construction of p53 deletion glioma cell model:the wild type p53(Wtp53)gene of glioma cells(U87MG and A172)was knockout by CRISPR-Cas9 gene editing technique and verified by knockout in three aspects:(1)p53 gene sequencing,(2)p53 gene expression verification,(3)p53 functional inactivation by detecting p53 downstream functional target gene p21 protein.3.Construction of p53 mutant glioma cell model:p53 lentivirus expression vector(Lvp53Mt248 and Lvp53Mt175)with amino acid point mutation at position 248 and 175th of p53 gene was stably transferred into U87MG-p53KO and A172-p53KO cells and verified by mutation in two aspects:(1)p53 gene expression was verified;(2)mutant p53 function was understood by detecting p53 downstream functional target gene p21 protein.4.Detection of MAGE-D4 expression:Total RNA and protein of U87MG and A172 cells with different p53 states were extracted,and the expression of MAGE-D4 mRNA and protein was detected by real-time fluorescence quantitative PCR(qRT-PCR)and Western imprinting(Western Blot).5.Determination of MAGE-D4 promoter activity:according to the results of bioinformatics analysis,the double luciferase reporter gene vector of MAGE-D4 core promoter containing or missing potential p53 binding sites was constructed and transfected into U87MG and A172 cells with different p53 states.the activity of MAGE-D4 promoter was understood by detecting luciferase activity.Results 1.MAGE-D4 core promoter region contains p53 binding site:bioinformatics analysis shows that there is a potential p53 binding site(59?83bp)in MAGE-D4 core promoter region(-358?172 bp).2.The glioma cell model with p53 deletion(U87MG-p53KO and A172-p53KO)was successfully constructed:after CRISPR-Cas9 knockout of p53wt in U87MG and A172,DNA sequencing confirmed that p53 gene had the expected deletion of p53 DNA fragment,the expression of p53 mRNA decreased significantly and p53 protein expression disappeared after U87MG knockout,and p53 mRNA and protein were almost not detected after A172 knockout.At the same time,with the knockout of p53 gene,the expression of p21 protein decreased,especially after U87MG knockout.3.The glioma cell model with p53 mutation(U87MG-p53Mt and A172-p53Mt)was successfully constructed.p53Mt248 and p53Mt175 lentivirus expression vectors were highly transfected into U87MG-p53KO and A172-p53KO cells,and the expression of p53 protein was significantly increased,and significant p53 protein expression band was observed;at the same time,the expression of p21 protein was increased.4.The effect of p53 on the expression of MAGE-D4 gene:compared with U87MG-p53Wt group,the expression of MAGE-D4 mRNA in U87MG-p53KO group,U87MG-p53Mt248 group and U87MG-p53Mt175 group was significantly higher than that in U87MG-p53Wt group,and the effect of U87MG-p53Mt248 and U87MG-p53Mt175 group on MAGE-D4 mRNA was better than that in U87MG-p53KO group.Compared with A172-p53wt group,the expression of MAGE-D4 mRNA in A172-p53Mt248 and A172-p53Mt175 group was significantly higher than that in A172-p53KO group,but there was no significant difference between A172-p53KO group and U87MG-p53Mt248 group.Western Blot results showed that the expression of MAGE-D4 protein in U87MG-p53KO group and U87MG-p53Mt248 group was higher than that in U87MG-p53Wt group,but the expression of MAGE-D4 protein in U87MG-p53Mt175 group had no significant change,and there was no significant difference in MAGE-D4 protein between U87MG-p53KO group and U87MG-p53Mt248 group.The level of MAGE-D4 protein in A172-p53Mt248 and A172-p53Mt175 group was significantly higher than that in A172-p53Wt group and A172-p53KO group,but there was no significant change in MAGE-D4 protein level in A172-p53KO group compared with A172-p53Wt group.5.Effect of p53 on the activity of MAGE-D4 promoter:the activity of MAGE-D4 core promoter reporter gene containing p53 binding site was determined.the results showed that the activity of MAGE-D4 promoter in U87MG-p53KO group,U87MG-p53Mt248 group and U87MG-p53Mt175 group was significantly higher than that in U87MG-p53KO group,U87MG-p53Mt248 group and U87MG-p53Mt175 group,and the activity of MAGE-D4 promoter in U87MG-p53Mt248 and U87MG-p53Mt175 group was significantly stronger than that in U87MG-p53KO group.A172-p53KO group.The activity of MAGE-D4 promoter in A172-p53Mt248 and A172-p53Mt175 group was significantly higher than that in A172-p53Wt group.However,the activity of MAGE-D4 promoter in A172-p53Mt248 and A172-p53Mt175 group was significantly higher than that in A172-p53KO group,and the activity of MAGE-D4 promoter in A172-p53Mt175 group was significantly higher than that in A172-p53KO group.The activity of MAGE-D4 core promoter reporter gene with further deletion of p53 binding site showed that there was no significant difference in the activity of MAGE-D4 core promoter between p53 Wt group and p53KO group in U87MG and A172.Conclusion The deletion of wild type p53 and amino acid point mutation at position 248 and 175th in gliomas can activate MAGE-D4 promoter and promote the expression of MAGE-D4.