Investigation and verification of the clinical significance and perspective of natural killer group 2 member D ligands in colon adenocarcinoma

Abstract

This study investigated and verified the diagnostic and prognostic values of natural killer group 2 member D ligand (NKG2DL) genes in colon adenocarcinoma (COAD). We downloaded NKG2DLs expression data and corresponding clinical parameters from The Cancer Genome Atlas (TCGA) and used bioinformatics techniques to investigate the values of NKG2DLs in COAD. Then, we used the GSE40967 cohort to verify the prognostic value of NKG2DLs. Finally, we verified the ULBP2 expression level in tissues, and also investigated the diagnostic and prognostic values of ULBP2 in COAD. The diagnostic receiver operating characteristic curves showed that ULBP1, ULBP2, ULBP3, and RAET1L had high diagnostic values in COAD [Area Under Curve (AUC) > 0.9]. In TCGA cohort, the univariate and multivariate survival analyses suggested that ULBP2 was correlated with the prognosis of COAD recurrence-free survival (RFS) and overall survival (OS). In GSE40967 cohort, ULBP2 was associated with CC RFS and OS. Reverse transcription-quantitative polymerase chain reaction and immunohistochemistry results showed that ULBP2 was highly expressed in COAD tumor tissues (P < 0.05) and both had diagnostic values (AUC > 0.7). Validated survival analysis showed that the high expression of ULBP2 had a worse prognosis in COAD OS and RFS. Thus, ULBP2 might be an independent diagnostic and prognostic biomarker of COAD.

Keywords: colon adenocarcinoma; diagnosis; natural killer group 2 member D ligands; prognosis; verification.

Figure 1

The differential expression and diagnostic ROC curves of NKG2DL genes in COAD. (A, B) The differential expression of NKG2DL genes in COAD: (A) Differential expression in the Edger and DESeq; (B) Expression distribution in TCGA; (C–J) The diagnostic ROC curves of NKG2DL genes in distinguish COAD tumor tissues and adjacent normal tissues in TCGA cohort: ROC curves of MICA (C); MICB (D); ULBP1 (E); ULBP2 (F); ULBP3 (G); RAET1E (H); RAET1G (I); RAET1L (J). Notes: COAD: colon adenocarcinoma; NKG2DL, Natural Killer Group 2 MemberD Ligand; ULBP: unique long 16 (UL16)-binding protein; MIC: Human Major Histocompatibility Complex (MHC) class I polypeptide-related sequence; TCGA: The Cancer Genome Atlas; NS: not significant; ROC: receiver operating characteristic; AUC: area under the curve; CI: confidence interval. * P<0.05; *** P<0.001; **** P<0.0001.

Figure 2

Kaplan–Meier survival curves for NKG2DL genes in COAD of TCGA cohort. Notes: RFS stratified by MICA (A); MICB (B); ULBP1 (C); ULBP2 (D); ULBP3 (E); RAET1E (F); RAET1G (G); RAET1L (H). OS stratified by MICA (I); MICB (J); ULBP1 (K); ULBP2 (L); ULBP3 (M); RAET1E (N); RAET1G (O); RAET1L (P). COAD: colon adenocarcinoma; NKG2DL, Natural Killer Group 2 Member D Ligand; ULBP: unique long 16 (UL16)-binding protein; MIC: Human Major Histocompatibility Complex (MHC) class I polypeptide-related sequence; TCGA: The Cancer Genome Atlas; RFS, recurrence-free survival; OS, overall survival.

Figure 3

Prognostic risk score model and time-dependent ROC curve of ULBP2 gene in COAD and CC RFS and OS based on TCGA cohort and GSE40967 cohort. (A, D) Risk score model plot including risk score ranking, survival status, and heatmaps in COAD in COAD RFS and OS; (B, E) Kaplan–Meier curves for low- and high-risk groups in COAD RFS and OS; (C, F) ROC curves for 1-, 2-, 3-, 4-, and 5-year survival rates from the risk score model in CC RFS and OS; (G and J) Risk score model plot including risk score ranking, survival status, and heatmaps in CC in CC RFS and OS; (H, K) Kaplan–Meier curves for low- and high-risk groups in CC RFS and OS; (I, L) ROC curves for 1-, 2-, 3-, 4-, and 5-year survival rates from the risk score model in CC RFS and OS. Notes: COAD: colon adenocarcinoma; CC: colon cancer; ULBP: unique long 16 (UL16)-binding protein; ROC, receiver operating characteristic; RFS, recurrence-free survival; OS, overall survival.

Figure 4

The relative expression and diagnostic ROC curve of ULBP2 gene in COAD tumor tissues and adjacent normal tissues. Notes: (A, B) The relative expression of ULBP2 gene; (C) diagnostic ROC curve. Notes: COAD: colon adenocarcinoma; ULBP: unique long 16 (UL16)-binding protein; AUC: area under the curve; CI: confidence interval.

Figure 5

Immunohistochemical staining analyses of ULBP2 expression in COAD tumor tissues and adjacent normal tissues. (A–D) ULBP2 staining results were observed in the cytoplasm of colon cells; (A) Positive staining of COAD tumor tissues; (B) Negative staining of COAD tumor tissues; (C) Positive staining of adjacent normal colon tissues; (D) Negative staining of adjacent normal tissues; (E, F) Diagnostic analysis of ULBP2 expression in COAD: (E) Scatter plot; (F) Diagnostic ROC curve; survival analysis of ULBP2 expression in COAD: (G, H) Kaplan–Meier survival curve of ULBP2 protein expression in COAD OS and RFS; (I, J) Stratified analysis of ULBP2 expression in different clinical parameter layers in COAD patients: (A) OS; (B) RFS. Notes: COAD, colon adenocarcinoma; ULBP: unique long 16 (UL16)-binding protein; ROC, receiver operating characteristic; AUC: area under the curve; CI: confidence interval; OS, overall survival; RFS, recurrence-free survival. Magnification, 200×.