Downregulation of NAT1 Expression is Associated with Poor Prognosis and Immune Infiltration in COAD

Figures & data

Table 1 Association Between NAT1 Expression and Clinicopathological Parameters in Patients with COAD

Characteristic	Low expression of NAT1	High expression of NAT1	P value
n	239	239
T stage, n (%)			0.010
T1	3 (0.6%)	8 (1.7%)
T2	31 (6.5%)	52 (10.9%)
T3	168 (35.2%)	155 (32.5%)
T4	37 (7.8%)	23 (4.8%)
N stage, n (%)			<0.001
N0	110 (23%)	174 (36.4%)
N1	66 (13.8%)	42 (8.8%)
N2	63 (13.2%)	23 (4.8%)
M stage, n (%)			0.003
M0	164 (39.5%)	185 (44.6%)
M1	45 (10.8%)	21 (5.1%)
Pathologic stage, n (%)			<0.001
Stage I	29 (6.2%)	52 (11.1%)
Stage II	73 (15.6%)	114 (24.4%)
Stage III	88 (18.8%)	45 (9.6%)
Stage IV	45 (9.6%)	21 (4.5%)
Primary therapy outcome, n (%)			0.045
PD	16 (6.4%)	9 (3.6%)
SD	3 (1.2%)	1 (0.4%)
PR	8 (3.2%)	5 (2%)
CR	86 (34.4%)	122 (48.8%)
Gender, n (%)			0.783
Female	115 (24.1%)	111 (23.2%)
Male	124 (25.9%)	128 (26.8%)
Age, n (%)			0.162
<=65	105 (22%)	89 (18.6%)
>65	134 (28%)	150 (31.4%)
Weight, n (%)			0.911
<=90	105 (38.5%)	84 (30.8%)
>90	48 (17.6%)	36 (13.2%)
Height, n (%)			0.789
<170	73 (28.5%)	54 (21.1%)
≥170	71 (27.7%)	58 (22.7%)
BMI, n (%)			0.517
<25	46 (18%)	41 (16%)
≥25	98 (38.3%)	71 (27.7%)
Residual tumor, n (%)			0.733
R0	169 (45.2%)	177 (47.3%)
R1	2 (0.5%)	2 (0.5%)
R2	14 (3.7%)	10 (2.7%)
CEA level, n (%)			0.006
<=5	87 (28.7%)	109 (36%)
>5	66 (21.8%)	41 (13.5%)
Perineural invasion, n (%)			0.115
NO	65 (35.9%)	70 (38.7%)
YES	29 (16%)	17 (9.4%)
Lymphatic invasion, n (%)			<0.001
NO	117 (27%)	149 (34.3%)
YES	102 (23.5%)	66 (15.2%)
Colon polyps present, n (%)			0.775
NO	92 (36.9%)	70 (28.1%)
YES	47 (18.9%)	40 (16.1%)
Age, median (IQR)	68 (55, 76)	69 (61, 78)	0.057

Figure 1 Expression levels of NAT1 mRNA in COAD from TIMER, TCGA and GEO databases. (A) Expression levels of NAT1 in different cancer types from TIMER database. (B) Expression of NAT1 mRNA was significantly down-regulated in COAD tumor tissues compared to normal colon tissues from the TCGA-COAD dataset. (C) Significantly decreased expression of NAT1 mRNA in COAD tumor tissues compared to adjacent paired normal colon tissues in the TCGA-COAD data. (D) GSE9348 dataset reveals a significantly lower NAT1 mRNA expression in COAD than in normal colon tissues. (E) GSE20916 dataset reveals a significantly lower NAT1 mRNA expression in COAD than in normal colon tissues.

Notes: *P<0.05, **P<0.01, ***P<0.001.

Table 2 Logistic Regression Analysis of the Relationship Between NAT1 Expression and Clinicopathological Parameters in COAD

Characteristics	Total(N)	Odds Ratio(OR)	P value
T stage (T3&T4 vs T1&T2)	477	0.492 (0.306–0.780)	0.003
N stage (N1&N2 vs N0)	478	0.319 (0.217–0.465)	<0.001
M stage (M1 vs M0)	415	0.414 (0.233–0.715)	0.002
Pathologic stage (Stage III&Stage IV vs Stage I&Stage II)	467	0.305 (0.207–0.446)	<0.001
Gender (Male vs Female)	478	1.069 (0.747–1.532)	0.714
Age (<=65 vs >65)	478	0.757 (0.524–1.091)	0.136
Weight (<=90 vs >90)	273	1.067 (0.636–1.799)	0.807
Height (<170 vs ≥170)	256	0.906 (0.552–1.484)	0.694
BMI (<25 vs ≥25)	256	1.230 (0.730–2.071)	0.435
Residual tumor (R1&R2 vs R0)	374	0.716 (0.322–1.551)	0.400
CEA level (<=5 vs >5)	303	2.017 (1.251–3.279)	0.004
Perineural invasion (YES vs NO)	181	0.544 (0.270–1.073)	0.083
Lymphatic invasion (YES vs NO)	434	0.508 (0.342–0.751)	<0.001
History of colon polyps (YES vs NO)	408	1.325 (0.883–1.993)	0.175

Figure 2 Expression levels of NAT1 in COAD clinical tissues. (A-D) Expression levels of NAT1 mRNA are analyzed using the TCGA-COAD dataset based on T, N, M, and pathological stages. (E) RT-qPCR analysis is conducted on COAD tissues and adjacent paired normal colon tissues. (F) Western blot (WB) analysis is conducted on COAD tissues and adjacent paired normal colon tissues.

Notes: **P<0.01; ***P<0.001.

Abbreviations: RT-qPCR, Quantitative reverse transcription polymerase chain reaction.

Figure 3 Diagnostic and prognostic value of NAT1 in COAD. (A) Relationship between NAT1 expression and DFS in the GSE14333 dataset. (B-D) Relationship between NAT1 expression and DFS, DSS and OS in the GSE17536 dataset. (E-G) Association of low or high NAT1 expression with OS, DSS and PFI in patients with COAD in the TCGA-COAD dataset. (H-I) ROC analysis of NAT1 in COAD, with an AUC of 0.713 (95% CI = 0.578 to 0.848) for the GSE20916 dataset and 0.912 (95% CI = 0.864 to 0.959) for the TCGA-COAD dataset.

Abbreviations: OS, overall survival; DFS, disease free survival; DSS, disease specific survival; PFI, progress free interval; ROC, Receiver operating characteristic; AUC, area under the curve.

Table 3 Univariate and Multivariate Cox Regression Analyses of Clinicopathological Factors Associated with OS in Patients with COAD

Characteristics	Total(N)	Univariate analysis		Multivariate analysis
		Hazard ratio (95% CI)	P value	Hazard ratio (95% CI)	P value
T stage	476
T1&T2	94	Reference
T3&T4	382	3.072 (1.423–6.631)	0.004	7.412 (0.997–55.111)	0.050
N stage	477
N0	283	Reference
N1&N2	194	2.592 (1.743–3.855)	<0.001	0.275 (0.099–0.760)	0.013
M stage	414
M0	348	Reference
M1	66	4.193 (2.683–6.554)	<0.001	1.774 (0.893–3.525)	0.102
Pathologic stage	466
Stage I&Stage II	267	Reference
Stage III&Stage IV	199	2.947 (1.942–4.471)	<0.001	7.176 (2.079–24.775)	0.002
Gender	477
Female	226	Reference
Male	251	1.101 (0.746–1.625)	0.627
Age	477
>65	283	Reference
<=65	194	0.621 (0.406–0.951)	0.028	0.510 (0.283–0.919)	0.025
Residual tumor	373
R0	345	Reference
R1&R2	28	4.364 (2.401–7.930)	<0.001	1.792 (0.874–3.674)	0.111
NAT1	477
Low	238	Reference
High	239	0.618 (0.417–0.917)	0.017	0.689 (0.395–1.200)	0.188

Figure 4 GO and KEGG enrichment analysis of NAT1 gene. (A-C) GO enrichment analysis showing BP (biological process), CC (cellular component) and MF (molecular function) of genes co-expressed with NAT1. (D) KEGG enrichment analysis of genes co-expressed with NAT1.

Table 4 Results of Gene Set Enrichment Analysis (GSEA) for NAT1

Description	setSize	enrichmentScore	pvalue	p.adjust	qvalue
KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION	270	−0.5872	0.0009	0.027	0.0239
KEGG_OLFACTORY_TRANSDUCTION	370	−0.4878	0.0009	0.027	0.0239
REACTOME_OLFACTORY_SIGNALING_PATHWAY	376	−0.4903	0.0009	0.027	0.0239
REACTOME_BASE_EXCISION_REPAIR	90	−0.6251	0.001	0.0271	0.0239
KEGG_ECM_RECEPTOR_INTERACTION	83	−0.6015	0.001	0.0271	0.0239
BIOCARTA_D4GDI_PATHWAY	12	0.7536	0.0064	0.1257	0.1112
BIOCARTA_MHC_PATHWAY	12	0.7293	0.0064	0.1257	0.1112
BIOCARTA_CTL_PATHWAY	13	0.7304	0.0068	0.131	0.1159
BIOCARTA_IL17_PATHWAY	15	0.6281	0.0074	0.1335	0.1181

Figure 5 Enrichment plots from the Gene Set Enrichment Analysis (GSEA). NAT1 were significantly enriched several pathways including: (A) Neuroactive ligand-receptor Interaction, (B) Olfactory transduction, (C) Olfactory signaling pathway, (D) ECM receptor interaction, (E) Base excision repair, (F) D4GDI pathway, (G) MHC pathway, (H) CTL pathway, and (I) IL17 pathway.

Abbreviations: NES, Normalized Enrichment Scores; FDR, False Discovery Rate.

Figure 6 Association analysis of NAT1 gene expression with immune infiltration. (A) Association between NAT1 expression and 24 tumor-infiltrating lymphocytes. (B-G) Correlation of NAT1 expression with immune infiltration levels of (B) Th17 cells, (C) B cells, (D) T helper cells, (E) T cells, (F) Th2 cells, and (G) NK cells.