Construction of a multigenic diagnostic, prognostic, and immune infiltration model with methylation-associated regulators in esophageal squamous cell carcinoma

Abstract

Background: Methylation-related regulators may be involved in the prognostic prediction of esophageal squamous cell carcinoma (ESCC). Our study aimed to apply bioinformatics to screen methylation-related regulators for the construction of a prognostic model for patients with ESCC and to assess their diagnostic value and correlation with immune infiltration.

Methods: Prognosis-related genes were identified from The Cancer Genome Atlas (TCGA) database. Methylation-associated genes were filtered using the GeneCards database. We used the least absolute shrinkage and selection operator (LASSO) and Cox proportional hazards regression to identify the prognostic indicators. We applied the single-sample gene set enrichment analysis (ssGSEA) to clarify the relationship between prognostic indicators and immune infiltration in patients with ESCC (n=82).

Results: We constructed a prognostic model using methylation-related regulators homocysteine-inducible ER protein with ubiquitin-like domain 1 (HERPUD1), trans-2,3-enoyl-CoA reductase (TECR), melanoma antigen gene A11 (MAGEA11), and NOP2/Sun RNA methyltransferase 6 (NSUN6) to evaluate the prognosis of patients with ESCC. A higher prognostic risk score was associated with shorter overall survival (OS) in patients with ESCC [hazard ratio (HR) =5.77, 95% confidence interval (CI): 2.13-15.58; P<0.001]. Time-dependent area under the curve (AUC) analysis revealed that HERPUD1, TECR, MAGEA11, and NSUN6 had high prognostic predictive value at different time points. Furthermore, we found that the combined diagnostic model based on HERPUD1, TECR, MAGEA11, and NSUN6 had excellent diagnostic efficacy for ESCC (AUC =0.911; 95% CI: 0.888-0.935). Finally, the ssGSEA algorithm showed that HERPUD1 was significantly positively correlated with immune infiltration at both the cellular and genetic levels, while TECR showed a significant negative correlation with immune infiltration levels.

Conclusions: Our prognostic model, built with the methylation-related regulators HERPUD1, TECR, MAGEA11, and NSUN6, could effectively predict prognosis in patients with ESCC, enhance diagnostic efficacy, and reflect immune cell infiltration in their microenvironment. Our findings are hypothesis generating and larger confirmatory studies are needed to validate our results.

Keywords: Esophageal squamous cell carcinoma (ESCC); diagnosis; immune infiltration; methylation; prognosis.

Figure 1

Screening of prognostic factors in ESCC. (A) ESCC-related prognostic factors were selected from TCGA database, and then functional enrichment analysis of prognostic factors was performed via GO analysis. (B) Methylation-related prognostic factors were screened with the GeneCards database. (C) LASSO cross-validation curves and (D) LASSO coefficient path diagrams were drawn to identify the nonzero coefficients. ESCC, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas; GO, Gene Ontology; LASSO, least absolute shrinkage and selection operator.

Figure 2

The association of HERPUD1, TECR, MAGEA11, and NSUN6 with clinical parameters in patients with ESCC. The expression level of HERPUD1, TECR, MAGEA11, and NSUN6 was used to analyze their correlation with clinical stage, pathological stage, histological grade, primary therapy outcomes, alcohol history, and reflux history. *, P<0.05. HERPUD1, homocysteine-inducible ER protein with ubiquitin-like domain 1; TECR, trans-2,3-enoyl-CoA reductase; MAGEA11, melanoma antigen gene A11; NSUN6, NOP2/Sun RNA methyltransferase 6; TPM, transcripts per million; ESCC, esophageal squamous cell carcinoma.

Figure 3

Verification HERPUD1, TECR, MAGEA11, and NSUN6 expression with the GEO and the TCGA databases in patients with ESCC. (A) HERPUD1, TECR, MAGEA11, and NSUN6 expression levels were evaluated with TCGA database. (B) GEO database was used to validate the expression levels of HERPUD1, TECR, MAGEA11, and NSUN6 in ESCC tissues with three GEO datasets: GSE161533, GSE45670, and GSE20347. ***, P<0.001. TPM, transcripts per million; HERPUD1, homocysteine-inducible ER protein with ubiquitin-like domain 1; TECR, trans-2,3-enoyl-CoA reductase; MAGEA11, melanoma antigen gene A11; NSUN6, NOP2/Sun RNA methyltransferase 6; GEO, Gene Expression Omnibus; TCGA, The Cancer Genome Atlas; ESCC, esophageal squamous cell carcinoma.

Figure 4

The association of HERPUD1, TECR, MAGEA11, and NSUN6 with OS, DSS, and PFS in patients with ESCC. The correlation of HERPUD1, TECR, MAGEA11, and NSUN6 with OS, DSS, and PFS in patients with ESCC was evaluated using Kaplan-Meier survival analysis. HR, hazard ratio; HERPUD1, homocysteine-inducible ER protein with ubiquitin-like domain 1; TECR, trans-2,3-enoyl-CoA reductase; MAGEA11, melanoma antigen gene A11; NSUN6, NOP2/Sun RNA methyltransferase 6; OS, overall survival; DSS, disease-specific survival; PFS, progression-free survival; ESCC, esophageal squamous cell carcinoma; CI, confidence interval.

Figure 5

The establishment of a prognostic nomogram model with HERPUD1, TECR, MAGEA11, and NSUN6 in patients with ESCC. (A) Time-dependent AUCs were used to evaluate the prognostic values of HERPUD1, TECR, MAGEA11, and NSUN6 at various time points. HERPUD1, TECR, MAGEA11, and NSUN6 were used to establish the (B) nomogram model and (C) calibration curves for patients with ESCC. HERPUD1, homocysteine-inducible ER protein with ubiquitin-like domain 1; TECR, trans-2,3-enoyl-CoA reductase; MAGEA11, melanoma antigen gene A11; NSUN6, NOP2/Sun RNA methyltransferase 6; AUC, area under the curve; ESCC, esophageal squamous cell carcinoma.

Figure 6

Risk scoring model of HERPUD1, TECR, MAGEA11, and NSUN6 in patients with ESCC. (A) Risk factor graph of the trends of prognostic factors and risk scores. (B) The relationship of risk score with OS was analyzed using a Kaplan-Meier plotter. “0” represents survivor; “1” represents death. HR, hazard ratio; CI, confidence interval; HERPUD1, homocysteine-inducible ER protein with ubiquitin-like domain 1; TECR, trans-2,3-enoyl-CoA reductase; MAGEA11, melanoma antigen gene A11; NSUN6, NOP2/Sun RNA methyltransferase 6; ESCC, esophageal squamous cell carcinoma; OS, overall survival.

Figure 7

The diagnostic efficacy of HERPUD1, TECR, MAGEA11, and NSUN6 in patients with ESCC. The ability of biomarkers (A) HERPUD1, (B) TECR, (C) MAGEA11, (D) NSUN6, and (E) a combined model in diagnosing ESCC as the ROC AUC. HERPUD1, homocysteine-inducible ER protein with ubiquitin-like domain 1; TECR, trans-2,3-enoyl-CoA reductase; MAGEA11, melanoma antigen gene A11; NSUN6, NOP2/Sun RNA methyltransferase 6; TPR, true positive rate; FPR, false positive rate; AUC, area under the curve; CI, confidence interval; ESCC, esophageal squamous cell carcinoma; ROC, receiver operator characteristic.

Figure 8

Correlation of HERPUD1, TECR, MAGEA11, and NSUN6 with immune infiltration in patients with ESCC. (A) The ssGSEA algorithm was used to calculate the relationship of HERPUD1, TECR, MAGEA11, and NSUN6 with the enrichment scores of 24 types of immune cells. The correlation of HERPUD1, TECR, MAGEA11, and NSUN6 with (B) immunoinhibitors, (C) MHC molecules, and (D) immunostimulators was measured with Spearman correlation analysis. *, P<0.05. HERPUD1, homocysteine-inducible ER protein with ubiquitin-like domain 1; TECR, trans-2,3-enoyl-CoA reductase; MAGEA11, melanoma antigen gene A11; NSUN6, NOP2/Sun RNA methyltransferase 6; MHC, major histocompatibility complex; ESCC, esophageal squamous cell carcinoma; ssGSEA, single-sample gene set enrichment analysis; Cor, correlation.

Figure 9

GO and KEGG analysis of HERPUD1 and TECR-related immune infiltration pathway enrichment in patients with ESCC. (A) Venn diagram represents the gene intersection of immune infiltration-related genes and HERPUD1-related DEGs; (B) GO and KEGG analysis of the gene intersection of immune infiltration-related genes and HERPUD1-related DEGs; (C) Venn diagram represents the gene intersection of immune infiltration-related genes and TECR-related DEGs; (D) GO and KEGG analysis of the gene intersection of immune infiltration-related genes and TECR-related DEGs. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; HERPUD1, homocysteine-inducible ER protein with ubiquitin-like domain 1; TECR, trans-2,3-enoyl-CoA reductase; DEGs, differentially expressed genes; BP, biological process; CC, cellular component; MF, molecular function; adj, adjusted.