TNC upregulation promotes glioma tumourigenesis through TDG-mediated active DNA demethylation

Abstract

Gliomas represent the most predominant primary malignant tumor in central nervous system. Thymine DNA glycosylase (TDG) is a central component in active DNA demethylation. However, the specific mechanisms of TDG-mediated active DNA demethylation in gliomas remain unclear. This research indicates TDG expression is overexpressed in gliomas and correlated with poor prognosis. TDG knockdown suppressed the malignant phenotype of gliomas both in vitro and vivo. Notably, RNA-seq analysis revealed a strong association between TDG and tenascin-C (TNC). ChIP-qPCR and MeDIP-qPCR assays were undertaken to confirm that TDG participates in TNC active DNA demethylation process, revealing decreased DNA methylation levels and elevated TNC expression as a result. Silencing TNC expression also suppressed the tumor malignant phenotype in both in vitro and in vivo experiments. Additionally, simultaneous silencing of TNC reduced or even reversed the glioma promotion caused by TDG overexpression. Based on our findings, we conclude that TDG exerts an indispensable role in TNC active DNA demethylation in gliomas. The DNA demethylation process leads to alternations in TNC methylation levels and promotes its expression, thereby contributing to the development of gliomas. These results suggest a novel epigenetic therapeutic strategy targeting active DNA demethylation in gliomas.

Fig. 1. Upregulation of TDG expression in glioma tissue associated with glioma grade and poor outcome.

A, B TDG mRNA expression in gliomas with normal brain tissues and different WHO grades from the TCGA database. C TDG mRNA expression levels of different histological subtypes were compared in gliomas from the TCGA database. D Representative IHC images of TDG in gliomas with different histological grades and NBT (200 µm). E The mRNA expression of TDG in SVGP12 and U251 cell lines were detected by RT-qPCR. F Kaplan-Meier survival curve of patients with glioma from the TCGA database stratified by TDG expression. G The receiver operator characteristic curve analysis of TDG utilizing the TCGA database.

Fig. 2. shTDG suppressed the cell proliferation, migration, invasion, and cell cycle progression of GBM cells in vivo and in vitro.

A Differential genes GO and KEGG pathway analysis based on TCGA RNA-seq. B The mRNA and protein levels to detect cell transfection efficiency. C CCK8 assays show cell growth in U251 and T98G cell lines infected with shCtrl and shTDG. D Transwell assays were used to detect the invasive ability of U251 and T98G cell lines infected with shCtrl and shTDG. E, F Cell migration was measured by a wound healing assay. G, H Flow cytometry analysis for cell cycle distribution in U251 and T98G cell lines infected with shCtrl and shTDG. I, J shCtrl and shTDG infected U251 cells were injected into nude mice. The tumors weight and volume in mice are shown to have undergone alteration. The representative IHC images of xenograft tumors are shown.

Fig. 3. TDG collaborates with TNC to facilitate active DNA demethylation.

A DEGs were identified in TDG knockdown or negative control stably transfected U251 cells and PCA. B DEGs heatmap. C, D GO and KEGG pathway analysis and GSEA based on DEGs from RNA-seq. E TNC DNA methylation changes and sites in the TCGA and GEO databases. F ChIP assays were performed on the TDG and TNC binding regions. G TNC 5mC changes in paraneoplastic and glioma tissues measured by MeDIP-qPCR. H The inhibition efficiency of TNC expression alteration was detected by RT-qPCR and western blotting. TNC 5mC changes in TDG knockdown and control U251 cells measured by MeDIP-qPCR. I TNC methylation levels in relation to mRNA expression levels from TCGA database. J Relationship between methylation level of TNC at chr9:117826025 and survival probability by Kaplan-Meier curve. K The correlation between TDG and TNC from TCGA database.

Fig. 4. TNC upregulation is linked to the tumor immune microenvironment and poor outcomes in glioma.

A, B TNC mRNA expression in gliomas with normal brain tissues and different WHO grades from the TCGA database. C The mRNA expression of TNC in SVGP12, U251 and T98G cell lines were detected by RT-qPCR. D Representative IHC images of TNC in gliomas with different histological grades and NBT (200 µm). E Univariate and multivariate Cox proportional hazards model using TCGA database. F Kaplan-Meier survival curve of patients with glioma from the TCGA database stratified by TNC expression. G Prognostic nomogram to predict 1-, 3-, and 5-year survival probability for patients with gliomas. H, I Correlation between TNC expression and 24 kinds of immune infiltration cells in TCGA database. The risk score was significantly correlated with the immune scores, stromal scores and ESTIMATE scores, respectively.

Fig. 5. TNC promotes GBM cell proliferation, migration, invasion, and cycle progression in vivo and in vitro.

A, B TNC-siRNA effectively inhibits the mRNA and protein expression of TNC in U251 and T98G cells. C CCK8 assays show cell growth in U251 and T98G cell lines infected with siCtrl and siTNC. D Cell migration was measured by a wound healing assay. E Transwell assays were used to detect the invasive ability of U251 and T98G cell lines infected with siCtrl and siTNC. F Flow cytometry analysis for cell cycle distribution in U251 and T98G cell lines infected with siCtrl and siTNC. G Tumor weight and volume alteration in mice. The representative IHC images of xenograft tumors are shown.

Fig. 6. TNC partially reversed the effect of TDG in GBM cells.

A, B The overexpression efficiency of TDG and TNC expression alteration was detected by RT-qPCR and western blotting. C CCK8 assay showing cell growth in U251 cells infected with TDG overexpression or the control (vector), and infected with siCtrl or siTNC. D, E Cell migration was measured by a wound healing assay. F, G Transwell assays were used to detect invasive ability in U251 cell lines.

Fig. 7. The epigenetic regulation of TNC expression in glioma.

The expression of TNC is increased by TDG-mediated active DNA demethylation, which is a crucial process in the development of the malignant phenotype of glioma.