Potential role of TWIST1 and its methylation in bladder urothelial carcinoma

Abstract

Background: Bladder urothelial carcinoma (BLCA), like other cancers, is strongly associated with genetic and epigenetic changes. TWIST1 is an epithelial-mesenchymal transition (EMT) promoter that has been linked to the development of many malignancies. It is still unclear, however, what role TWIST1 plays in BLCA, and the relationship between TWIST1 transcript levels and its promoter methylation and immune infiltration has been reported even less. This study aimed to reveal the potential role of TWIST1 promoter methylation-related changes in BLCA.

Methods: Transcriptional expression data of TWIST1 in BLCA were acquired from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and University of Alabama at Birmingham (UALCAN) databases. TWIST1 methylation levels and prognosis were sourced from Gene Expression Profiling Interactive Analysis 2 (GEPIA2), LinkedOmics, cBio Cancer Genomics Portal (c-BioPortal), MethSurv, and DNA Methylation Information Visualization Database (DNMIVD) databases. The methylation status of the BLCA-associated TWIST1 in preoperative and postoperative urinary exfoliated cells was subsequently analyzed using methylation-specific real-time fluorescence polymerase chain reaction, with validation of accuracy through pyrophosphate sequencing. Finally, from the Gene Set Cancer Analysis (GSCA) and Tumor-Immune System Interaction Database (TISIDB) databases, we obtained the association between TWIST1 transcript expression and DNA methylation and cancer immune infiltration and immunolabelling.

Results: Our study demonstrated that TWIST1 expression was down-regulated in BLCA, which was negatively correlated with DNA methylation. The association between TWIST1 promoter hypermethylation and the progression, staging, grading, and recurrence of BLCA is highly significant. Furthermore, we revealed that hypermethylation of both the preoperative and postoperative TWIST1 promoters is useful as a biomarker for monitoring BLCA recurrence, particularly when considering the methylation status of specific CpG sites. Additionally, we observed that TWIST1 expression, promoter methylation, and immune infiltration immunoreactive markers correlated significantly in BLCA.

Conclusions: We propose that TWIST1 holds great promise as a diagnostic and therapeutic target for BLCA, with the potential to influence tumor progression and patient prognosis through the regulation of immune cell infiltration. We hope these findings contribute valuable insights to the field of BLCA research.

Keywords: Bladder urothelial carcinoma (BLCA); TWIST1; epigenetics; methylation; prognosis.

Figure 1

TWIST1 expression is down-regulated in BLCA. (A) TWIST1 expression levels in normal and tumor tissues in TCGA database, obtained from R package ggplot2 analysis. (B) TWIST1 expression levels in normal and tumor tissues in TCGA database, obtained from the UALCAN database analysis. (C,D) TWIST1 expression levels in normal and tumor tissues in GSE13507 and GSE37815. (E-J) Boxplot showing TWIST1 expression in subgroup analyses: stage (E), age (F), gender (G), race (H), weight (I), smoking habit (J). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, P≥0.05. BLCA, bladder urothelial carcinoma; TCGA, The Cancer Genome Atlas; UALCAN, University of Alabama at Birmingham.

Figure 2

Prognostic analysis of TWIST1 gene characterization. (A) Unifactorial Cox regression analyses. (B) Multifactorial Cox regression analyses. (C) Kaplan-Meier survival curves for the TWIST1. The HR was employed to compare the relatives of the low-expression sample to those of the high-expression sample. HR >1 suggest a gene is a risk factor, while HR <1 shows that it is not. HR =1 indicates that the gene is neither a risk factor nor a protective factor. Additionally, the HR (95% CI) was used to determine the LT₅₀ for various groups. (D) ROC curves and AUC values of genes at various times, the higher the AUC value indicates, the better the model predicts, 0.5 is considered that the model is close to random and meaningless, usually the AUC of prognostic model should be above 0.7. CI, confidence interval; pTNM, postsurgical tumor-node-metastasis; HR, hazard ratio; AUC, area under the ROC curve; ROC, receiver operating characteristic; LT₅₀, median survival time.

Figure 3

Co-expression network of TWIST1 in BLCA. (A) Volcano map of TWIST1 related genes in BLCA (red dots: these represent genes that are significantly positively correlated with TWIST1; green dots: these indicate genes that are significantly negatively correlated with TWIST1). (B,C) Top 50 BLCA genes that have positive (B) and negative (C) relationships with TWIST1 are displayed in a heat map. (D) TWIST1 co-expressed gene enrichment study using GO and KEGG. FDR, false discovery rate; BLCA, bladder urothelial carcinoma; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

TWIST1 promoter methylation levels are upregulated in BLCA expression. (A) Using UALCAN database, we analyzed methylation levels at the TWIST1 promoter in patients with BLCA from the TCGA. (B-G) TWIST1 methylation level in BLCA patients obtained from the UALCAN database was examined in relation to various factors, including cancer stage (B), smoking status (C), patient’s age (D), race (E), gender (F), and patient’s weight (G). ****, P<0.0001; ns, P≥0.05. BLCA, bladder urothelial carcinoma; UALCAN, University of Alabama at Birmingham; TCGA, The Cancer Genome Atlas.

Figure 5

Correlation analysis between TWIST1 methylation and prognosis. (A) H&E: pathological sections of a patient with preoperative and postoperative TWIST1 hypermethylation and recurrence (left: 100×; right: 200×; No. 1365). (B) Kaplan-Meier survival curves. (C) MethSurv was used to determine TWIST1 DNA methylation. (D) A correlation study of TWIST1 expression and methylation in BLCA patients. mRNA, messenger RNA; H&E, hematoxylin and eosin; BLCA, bladder urothelial carcinoma.

Figure 6

TWIST1 and its DNA methylation are linked to immune infiltration. (A) Correlation between TWIST1 expression and immune cells. (B) Correlation between TWIST1 methylation and immune cells. mRNA, messenger RNA; FDR, false discovery rate; DC, dendritic cell.

Figure 7

Correlation of TWIST1 transcript expression/DNA methylation with immunomarkers. (A) Survival heatmap of the top 20 in BLCA immunomarkers associated with TWIST1. Higher and lesser risk are represented by red and blue squares, accordingly. Bordered rectangles in the prognosis analysis show significant negative and positive findings (P<0.05). (B) Spearman’s correlation between TWIST1 expression and Treg, Th1, TNFSF4 and IL10. (C) Spearman’s correlation between TWIST1 methylation and Treg, Th1, TNFSF4, and IL10. HR, hazard ratio; Treg, regulatory T; BLCA, bladder urothelial carcinoma; Th1, T helper 1; TNFSF4, tumor necrosis factor ligand superfamily member 4; IL10, interleukin 10.