Liu Chang, Ge Yingying, Luo Bin, Xie Xiaoxun, Shen Ning, Nong Weixia, Bi Shuiqing, Lin Lina, Wei Xing, Wu Song, Xiao Shaowen, Zhang Qingmei
Department of Neurosurgery, The First Affiliated Hospital of Guangxi Medical University Nanning, Guangxi, P. R. China.
Department of Histology and Embryology, School of Pre-Clinical Medicine, Guangxi Medical University Nanning, Guangxi, P. R. China.
Am J Transl Res. 2021 Apr 15;13(4):2241-2255. eCollection 2021.
The family of MAGE genes is well known due to the majority of MAGE genes expressing specifically in tumor tissues while restrictedly in normal tissues. MAGE-D4 is one of the MAGE family and considered as a promising target for glioma immunotherapy because of its overexpression in glioma and restricted expression in normal tissues. Whereas the mechanism of MAGE-D4 heterogeneous expression in glioma has not yet been elucidated. In this study, the transcriptional regulation mechanism of MAGE-D4 in glioma is focused from the perspectives of promoter methylation and SP1.
Dual-luciferase reporter assay was performed to identify the core promoter of MAGE-D4 gene. Mass spectrometry was applied to quantify the methylation status of MAGE-D4 promoter in 50 glioma and 9 normal brain tissues. The influence of methylation and SP1 on MAGE-D4 transcriptional activity was evaluated by dual-luciferase reporter assay, qRT-PCR, western blot and ChIP-qPCR. Decitabine, an epigenetic drug, was used to treat the glioma cells. Then the treated cells were evaluated the influence of demethylation on SP1 binding to MAGE-D4 promoter.
The -358 to +172 bp region was identified as the core promoter of MAGE-D4 gene which demonstrated hypomethylated and negative correlation between methylation level and MAGE-D4 mRNA expression in glioma tissues. For single CpG unit analysis, 8 CpG units (CpG unit 1, 2, 3, 4, 5, 6, 9 and 12) in MAGE-D4 core promoter showed hypomethylated in glioma and the methylation level of CpG unit 6 was positively associated with the prognosis of glioma patients. Furthermore, the methylation level of CpG unit 1 and 6 was negative negatively correlated with MAGE-D4 mRNA expression. Then, the results demonstrated that the promoter activity of MAGE-D4 was decreased by methylation in glioma cell lines. In addition, SP1 can binds directly to the MAGE-D4 promoter leading to up-regulation of MAGE-D4 mRNA through activation of its promoter. Finally, demethylation of MAGE-D4 promoter could benefit the SP1 binding and resulting co-activation of MAGE-D4 promoter by demethylation and SP1 in glioma cell lines.
These findings indicate that the synergies of promoter hypomethylation and SP1 up-regulated MAGE-D4 transcription in glioma, which implies a potential approach to resolve the heterogeneous expression of MAGE-D4 in order to establish foundation for the MAGE-D4 based glioma therapy.
MAGE基因家族广为人知，因为大多数MAGE基因在肿瘤组织中特异性表达，而在正常组织中表达受限。MAGE-D4是MAGE家族成员之一，因其在胶质瘤中过表达而在正常组织中表达受限，被认为是胶质瘤免疫治疗的一个有前景的靶点。然而，MAGE-D4在胶质瘤中异质性表达的机制尚未阐明。在本研究中，从启动子甲基化和SP1的角度聚焦于胶质瘤中MAGE-D4的转录调控机制。
采用双荧光素酶报告基因测定法鉴定MAGE-D4基因的核心启动子。应用质谱法对50例胶质瘤组织和9例正常脑组织中MAGE-D4启动子的甲基化状态进行定量分析。通过双荧光素酶报告基因测定法、qRT-PCR、蛋白质印迹法和ChIP-qPCR评估甲基化和SP1对MAGE-D4转录活性的影响。使用表观遗传药物地西他滨处理胶质瘤细胞。然后评估处理后的细胞去甲基化对SP1与MAGE-D4启动子结合的影响。
-358至+172 bp区域被鉴定为MAGE-D4基因的核心启动子，该区域在胶质瘤组织中表现为低甲基化，且甲基化水平与MAGE-D4 mRNA表达呈负相关。对于单个CpG单元分析，MAGE-D4核心启动子中的8个CpG单元（CpG单元1、2、3、4、5、6、9和12）在胶质瘤中表现为低甲基化，且CpG单元6的甲基化水平与胶质瘤患者的预后呈正相关。此外，CpG单元1和6的甲基化水平与MAGE-D4 mRNA表达呈负相关。然后，结果表明胶质瘤细胞系中甲基化可降低MAGE-D4的启动子活性。此外，SP1可直接与MAGE-D4启动子结合，通过激活其启动子导致MAGE-D4 mRNA上调。最后，MAGE-D4启动子的去甲基化有利于SP1结合，并导致胶质瘤细胞系中去甲基化和SP1对MAGE-D4启动子的协同激活。
这些发现表明启动子低甲基化和SP1协同上调胶质瘤中MAGE-D4的转录，这意味着一种解决MAGE-D4异质性表达的潜在方法，以便为基于MAGE-D4的胶质瘤治疗奠定基础。
