Melanoma-associated antigen (MAGE) family genes have been considered as potentially promising targets for anticancer immunotherapy. MAGED4 was originally identified as a glioma-specific antigen. Current knowledge about MAGED4 expression in glioma is only based on mRNA analysis and MAGED4 protein expression has not been elucidated. In the present study, we investigated this point and found that MAGED4 mRNA and protein were absent or very lowly expressed in various normal tissues and glioma cell line SHG44, but overexpressed in glioma cell lines A172,U251,U87-MG as well as glioma tissues, with significant heterogeneity. Furthermore, MAGED4 protein expression was positively correlated with the glioma type and grade. We also found that the expression of MAGED4 inversely correlated with the overall methylation status of the MAGED4 promoter CpG island. Furthermore, when SHG44 and A172 with higher methylation were treated with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR) reactivation of MAGED4 mRNA was mediated by significant demethylation in SHG44 instead of A172. However, 5-AZA-CdR treatment had no effect on MAGED4 protein in both SHG44 and A172 cells. In conclusion, MAGED4 is frequently and highly expressed in glioma and is partly regulated by DNA methylation. The results suggest that MAGED4 might be a promising target for glioma immunotherapy combined with 5-AZA-CdR to enhance its expression and eliminate intratumor heterogeneity.

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ObjectiveThe study evaluated the efficacy of combined epigenetic drugs of decitabine (DAC), valproic acid (VPA), and trichostatin A (TSA) on immunotherapy against glioma.MethodsThe expression and prognosis of MAGE-D4 in glioma were analyzed online, and the expression of MAGE-D4 and HLA-A2 in glioma induced by epigenetic drugs was detected by qRT-PCR, Western blot, and flow cytometry. The methylation status of the MAGE-D4 promoter was determined by pyrosequencing. An HLA-A2 restricted MAGE-D4 peptide was predicted and synthesized. An affinity assay and a peptide/HLA complex stability assay were performed to determine the affinity between peptide and HLA. CCK8 assay, CFSE assay, ELISA and ELISPOT were performed to detect the function of MAGE-D4 peptide-specific T cells. Flow cytometry, ELISA, and cytotoxicity assays were used to detect the cytotoxicity effect of MAGE-D4 peptide-specific T cells combined with epigenetic drugs against glioma in vitro. Finally, the glioma-loaded mouse model was applied to test the inhibitory effect of specific T cells on gliomas in vivo.ResultsMAGE-D4 was highly expressed in glioma and correlated with poor prognosis. Glioma cells could be induced to express MAGE-D4 and HLA-A2 by epigenetic drugs. MAGE-D4-associated peptides were found that induce DCs to stimulate the highest T-cell activities of proliferation, IL-2 excretion, and IFN-γ secretion. MAGE-D4 peptide-specific T cells treated with TSA only or combining TSA and DAC had the most cytotoxicity effect, and its cytotoxicity effect on glioma cells decreased significantly after HLA blocking. In vivo experiments also confirmed that MAGE-D4-specific T cells inhibit TSA-treated glioma.ConclusionMAGE-D4 is highly expressed in glioma and correlated with the prognosis of glioma. The novel MAGE-D4 peptide identified was capable of inducing MAGE-D4-specific T cells that can effectively inhibit glioma growth, and the epigenetic drug application can enhance this inhibition.

Regulatory ability of micro-ribose nucleic acid-130a (miRNA-130a) in the proliferation and invasive growth of human brain glioma cells and its mechanism were investigated. RT-qPCR was used to analyze expression of miRNA-130a in U-87MG glioma specimens; lipidosome was used to mediate miRNA-130a mimic transfecting glioma cells and the expression of miRNA-130a was detected by using RT-qPCR after transfection; methyl thiazolyl tetrazolium (MTT) assay and flow cytometry (FCM) were adopted to evaluate the changes in biological characteristics of cell growth and proliferation; the migration and invasion abilities of tumor cells were measured through scratch assay and Transwell in vitro cell migration assay. In miRNA-130a mimic-transfected U-87MG cells, RT-qPCR showed that the expression of miRNA-130a was upregulated; MTT assay and FCM revealed that the cell growth was strengthened; scratch assay and Transwell in vitro cell migration assay verified that the migration and invasion abilities of cells were enhanced. In conclusion, the high expression of miRNA-130a can promote growth and invasion, indicating that miRNA-130a can be considered as a candidate target of gene therapy for glioma.