Transcriptional Regulation Of MAGE-D4 In Glioma: The Role Of Promoter Methylation And Transcription Factor Sp1

Objective:Discuss influences of methylation and transcription factor Sp1 on the activity of MAGE-D4 promoter.Methods:1.Bioinformatics analysis: According to the public database,inquire the region of MAGE-D4 promoter and transcription factors binding sites.Illustrate the expression characteristics of MAGE-D4 and Sp1 and their correlation in glioma.2.Mass Array: DNA of 50 Gliomas and 9 normal brain samples are extracted.Mass Array is performed to detect the methylation level of MAGE-D4 promoter.3.Construct luciferase reporter gene vectors: According to the result of bioinformatics analysis,five reporter gene vectors of truncated MAGE-D4promoters(pGL3-P1~P3)are designed.4.Luciferase assay: pGL3-P1~P5 are transfecting into cell lines to detect luciferase activity and then determine the core promoter of MAGE-D4.The sifted pGL3-P is used in the follow-up experiments.5.qRT-PCR: Total RNA is extracted to detect the mRNA expression ofMAGE-D4 and Sp1 in the glioma cell lines by qRT-PCR.6.Western Blot: Nuclear protein is extracted to detect the protein expression of Sp1 in the glioma cell lines by Western Blot.7.ChIP-qPCR: Compound of DNA and Sp1 protein is enriched by Sp1 antibody,and qRT-PCR is performed to detect the Sp1 and MAGE-D4 promoter sequence are combined.Results:1.According to the bioinformatics analysis,MAGE-D4 promoter sequences have many binding sites for transcription factors like Sp1,and there are four potential binding sites for Sp1 in MAGE-D4 core promoter region.In addition,there is certain co-expression correlation between MAGE-D4 and Sp1 in glioma,and their expressions are higher in glioma than in normal tissue.Moreover,some binding sites of Sp1 overlap with methylation sites in core promoter region of MAGE-D4.Hence,this article choose transcription factor Sp1 to further study its transcriptional regulation impact on MAGE-D4.2.Five reporter gene vectors of truncated MAGE-D4 promoters were designed.Results of Luciferase assay showed that the activity of-358~+172 bp truncated promoter was the highest.Therefore,in this paper,MAGE-D4 core promoter sequences are believed to be located in this region.3.According to the Mass Array results of glioma samples,the CpG islands in the MAGE-D4 promoter region showed a lower overall methylation level.4.According to results of Luciferase assay,methylated modification could reduce the activity of MAGE-D4 promoter.5.Results of Luciferase assay and qRT-PCR showed that transcription factor Sp1 could enhance the activity of MAGE-D4 promoter and promotetranscription.6.Results of ChIP-qPCR indicated that transcription factor Sp1 could bind to MAGE-D4 promoter region directly,which showed that Sp1 regulated MAGE-D4 transcription by directly affecting MAGE-D4 promoter.And DAC treating glioma cell lines can improve the binding ability of Sp1 to the MAGE-D4 promoter.7.In addition,reporter gene vector which was transfected by pcDNA-Sp1 without methylated modification and contained core promoter region of MAGE-D4,showed relatively highest activity.This indicated that there may be synergistic effects of Sp1 and demethylation modification enhancing the activity of MAGE-D4 promoter.Conclusion:1.The core promoter region of MAGE-D4 is located in-358~+172 bp.2.The activity of MAGE-D4 promoter is related to methylated modification.3.Transcription factor Sp1 could enhance the activity of MAGE-D4 promoter and promote transcription.4.There are synergistic effects of Sp1 and demethylated modification in enhancing the activity of MAGE-D4 promoter.