Bromodomain containing 4 transcriptionally activated Deltex E3 ubiquitin ligase 2 contributes to glioma progression and predicts an unfavorable prognosis

Abstract

Background: Glioblastoma multiforme (GBM) is the most common type of glioma, and the most aggressive brain malignancy in adults. This study sought to identify novel survival-status related markers, and examine their function in glioma.

Methods: The gene expression, survival heatmaps, and Kaplan-Meier survival plots of the genes were analyzed by using gene expression profiling interactive analysis (GEPIA) dataset, Linked Omics. The single-cell data analysis and tumor immune infiltration analysis was conducted by Tumor Immune Estimation Resource (TIMER) dataset. DBTRG and U251 cells with silenced Deltex E3 ubiquitin ligase 2 (DTX2) expression were constructed and used for Cell Counting Kit 8 (CCK-8), and wound healing assay in vitro. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis was used to explore the histone activation marks and transcription factors DTX2 promoter. Dual-luciferase assays were carried out to detect the luciferase activities of bromodomain containing 4 (BRD4) binding to DTX2.

Results: We first conducted a survival-status analysis to identify survival status-related genes in The Cancer Genome Atlas GBM and low-grade glioma data sets. A subsequent analysis identified 3 novel prognostic biomarkers; that is, DTX2, cytochrome P450 oxidoreductase, and Williams-Beuren syndrome chromosomal region 16 protein. In the validation Chinese Glioma Genome Atlas data sets, DTX2 showed the best performance, and was examined in a further analysis. Next, 3 short-hairpin ribonucleic acids were designed to silence DTX2 expression, and CCK-8 and wound-healing assays were applied to study the function of DTX2. We found that DTX2-silenced glioma cells exhibited a significant decrease in their growth and migration capabilities. Finally, the molecular basis for increased DTX2 in glioma was investigated via ChIP-Seq analysis and luciferase assays. The analysis revealed that DTX2 was transcriptionally activated by BRD4.

Conclusions: In conclusion, BRD4 transcriptionally activates DTX2, contributes to glioma progression, predicts an unfavorable prognosis, and could provide new options for glioma prognosis prediction and treatment.

Keywords: Deltex E3 ubiquitin ligase 2 (DTX2); Glioma; bromodomain containing 4 (BRD4); survival status.

Figure 1

Survival-status revealed prognosis-related genes in glioma. (A) Survival-status analysis showing the expression heatmaps of the top 50 positive and negative correlated genes in TCGA GBM data set; (B) survival-status analysis showing the expression heatmaps of the top 50 positive and negative correlated genes in TCGA LGG data set. GBM, glioma multiforme; LGG, low-grade glioma; TCGA, The Cancer Genome Atlas.

Figure 2

The survival and expression analysis revealed that DTX2 was a high-risk prognostic marker. (A) All 24 genes were found to be related to survival status in the LGG and GBM groups; (B) OS map showing the prognostic performance of the 24 common genes in the GBM and LGG data sets; (C) all 10 genes showed differential expression in TCGA GBM and LGG data sets; (D) Kaplan-Meier survival plots of the 10 genes in TCGA GBM and LGG data sets using GEPIA dataset (30). Genes with a higher expression and an unfavorable prognosis are labeled in red, and genes with a lower expression and favorable prognosis are labeled in blue. *, P<0.05. LGG, low-grade glioma; GBM, glioma multiforme; DTX2, Deltex E3 ubiquitin ligase 2; OS, overall survival; TCGA, The Cancer Genome Atlas.

Figure 3

DTX2 was highly expressed in malignant glioma cells. (A) DTX2 showed increased expression in the CGGA glioma data sets of higher grades, and the IDH WT and 1p/19q co-deletion groups; (B) Kaplan-Meier survival plots of DTX2 expression in the 3 CGGA array data sets; (C) expression of DTX2 in the single-cell RNA-Seq of adult and pediatric glioblastoma patients; (D) correlation analysis of DTX2 with tumor-infiltrating immune cells in GBM and LGG. TPM, transcripts per million; DTX2, Deltex E3 ubiquitin ligase 2; CGGA, Chinese Glioma Genome Atlas; IDH, isocitrate dehydrogenase; WT, wild type; LGG, low-grade glioma.

Figure 4

DTX2-silenced glioma cells showed decreased proliferation and migration capabilities. (A) The expression of DTX2 was examined by RT-qPCR in the control and 3 shRNA groups; (B) CCK8 assays showed the proliferation capability of the DTX2-silenced glioma cells; (C) wound-healing assays showed that the migration capability of the DTX2-knocked down cells were observed under electron microscope (magnification, 40×). All assays were replicated 3 times. *, P<0.05. DTX2, Deltex E3 ubiquitin ligase 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; shRNA, short-hairpin ribonucleic acid; RT-qPCR, real-time quantitative PCR; CCK8, Cell Counting Kit 8.

Figure 5

DTX2 is transcriptionally activated by BRD4 in glioma. (A) ChIP-sequencing peaks of histone activation marks and transcription factors were analyzed, and binding peaks within the DTX2 promoter are shown; (B) the expression correlation of DTX2 with transcription factors MAX, BRD4, and BRD2 in TCGA GBM and LGG data sets; (C) the expression correlation of DTX2 with transcription factors MAX, BRD4, and BRD2 in the CGGA data sets; (D) BRD4 inhibitor JQ1 repressed DTX2 expression in glioma cells; (E) dual-luciferase assays showing that JQ1 reduced the luciferase activities. *, P<0.05; **, P<0.01. TPM, transcripts per million; DTX2, Deltex E3 ubiquitin ligase 2; BRD4, bromodomain containing 4; WT, wild type; MUT, mutant; ChIP, Chromatin immunoprecipitation; TCGA, The Cancer Genome Atlas; GBM, glioma multiforme; LGG, low-grade glioma.