Exonuclease 1 (EXO1) is a Potential Prognostic Biomarker and Correlates with Immune Infiltrates in Lung Adenocarcinoma

Figures & data

Figure 1 Associations between EXO1 expression and clinicopathological features in LUAD. (A) EXO1 alterations, as determined by the cBioPortal, including amplification, missense mutation, and truncating mutation. (B) Pan-cancer analysis for expression levels of EXO1 in different types of cancers from the Oncomine. Different expression levels of EXO1 between tumor and normal tissues in LUAD, based on data from the TCGA database (C) and the GSE31210 (D). (E) Different promoter methylation levels of EXO1 in tumor and normal tissues in LUAD. (F) Different expression levels of EXO1 in TP53-mutant and TP53-wild-type groups in LUAD. **p<0.01, p < 0.05 was considered to be significant.

Abbreviations: LUAD, lung adenocarcinoma; TCGA, the Cancer Genome Atlas.

Table 1 Cox Regression Analysis for Overall Survival of LUAD Patients from the TCGA Database

Variable	Univariate	Multivariate
	P value	HR (95% CI)	P value
Age	0.259	1.014 (0.996–1.032)	0.128
Gender (female vs male)	0.688	1.068 (0.76–1.501)	0.705
T stage	< 0.001	1.38 (1.114–1.711)	0.003
N status	< 0.001	1.58 (1.281–1.949)	< 0.001
M status	0.007	1.479 (0.811–2.698)	0.202
TP53 mutation	0.179	1.134 (0.834–2.567)	0.315
EXO1 expression	< 0.001	1.075 (1.034–1.118)	< 0.001

Note: p < 0.05 was considered to be significant.

Abbreviations: LUAD, lung adenocarcinoma; TCGA, the Cancer Genome Atlas; HR, hazard ratio; CI, confidence interval.

Table 2 Cox Regression Analysis for Overall Survival of 78 LUAD Patients in Our Center

Variables	Univariate	Multivariate
	P value	HR (95% CI)	P value
Age	< 0.001	1.061(1.025–1.100)	0.001
Gender (female vs male)	0.315	0.654(0.338–1.268)	0.209
Stage	< 0.001	2.640(1.957–3.561)	< 0.001
EXO1 H-score	0.037	4.427(1.027–19.088)	0.046

Note: p < 0.05 was considered to be significant.

Abbreviations: LUAD, lung adenocarcinoma; HR, hazard ratio; CI, confidence interval.

Figure 2 High EXO1 level correlated with clinicopathological features and indicated poor prognosis in LUAD patients among public databases. Box plot showing EXO1 expression levels in tumors based on stage (A), nodal metastasis status (B) and distant metastasis status (C) determined by the UALCAN database. High mRNA expression of EXO1 was correlated with poor DFS (D) and OS (E) in LUAD patients from the TCGA database. Overexpression of EXO1 predicted poor OS in LUAD patients from GSE31210 (F). *p<0.05. p < 0.05 was considered to be significant.

Abbreviations: LUAD, lung adenocarcinoma; DFS, disease-free survival; OS, overall survival; TCGA, the Cancer Genome Atlas.

Figure 3 Expression and prognostic value of EXO1 in our cohort. (A) HE staining of EXO1 in tumor and matched normal tissue samples, Scale Bar for × 40 images, 50 μm. Positive staining (brown) of EXO1 was detected in tumoral epithelial cells, macrophages, and partially positive in vascular endothelial cells in tumor tissues. And positive staining of EXO1 in macrophages and partially positive in normal epithelial cells and vascular endothelial cells in normal tissues. EXO1 was negative in stromal cells in both tumor and normal tissues. (B) Immunohistochemistry scores showed that EXO1 was highly expressed in LUAD tumor tissue samples compared with normal tissue samples. (C) Kaplan-Meier survival analysis indicated that a high EXO1 immunohistochemistry score was associated with poor OS in LUAD patients. **p<0.01. p < 0.05 was considered to be significant.

Abbreviations: LUAD, lung adenocarcinoma; OS, overall survival.

Figure 4 KEGG and GSEA analyses of DEGs in LUAD patients. (A) Volcano plot of DEGs were identified between the EXO1 high and low expression groups. (B) Top 10 biological pathways according to KEGG analysis were shown. (C) Top 10 biological pathways enriched among the EXO1 upregulated DEGs through GSEA were shown. The overlapping pathways of the KEGG and GSEA analyses were Cell cycle (D), oocyte meiosis (E), and DNA replication (F). p < 0.05 was considered to be significant.

Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; DEGs, differentially expressed genes; NES, normalized enrichment score; FDR, false discovery rate.

Figure 5 The relationships of EXO1 with immune cells and the PPI network of EXO1. (A) The infiltrating levels of B cells and CD4 + T cells were negatively associated with EXO1 expression in LUAD patients. (B) Survival analysis showed that low levels of B cells and DCs predicted poor OS in LUAD. (C) The top 25 hub genes from the PPI network were shown. p < 0.05 was considered to be significant.

Abbreviations: PPI, protein-protein interaction; DCs, dendritic cells; OS, overall survival.

Table 3 Top 25 Hub Genes in PPI Network of EXO1

Name	Abbreviation	Function
Stratifin	SFN	Cell cycle checkpoint protein
Tryptophan 5-Monooxygenase Activation Protein Beta	YWHAB	Cell cycle checkpoint protein
Tryptophan 5-Monooxygenase Activation Protein Epsilon	YWHAE	Cell cycle checkpoint protein
Tryptophan 5-Monooxygenase Activation Protein Gamma	YWHAG	Cell cycle checkpoint protein
Tryptophan 5-Monooxygenase Activation Protein Eta	YWHAH	Cell cycle checkpoint protein
Tryptophan 5-Monooxygenase Activation Protein Theta	YWHAQ	Cell cycle checkpoint protein
Tryptophan 5-Monooxygenase Activation Protein Zeta	YWHAZ	Cell cycle checkpoint protein
Proliferating Cell Nuclear Antigen	PCNA	Cell cycle regulation; DNA replication
MutL Homolog 1	MLH1	MMR; DNA damage; Meiosis
BLM RecQ Like Helicase	BLM	DNA replication and repair
DNA Replication Helicase/Nuclease 2	DNA2	DNA replication and repair
ATM Serine/Threonine Kinase	ATM	DSBR; pre-B cell allelic exclusion
ATR Serine/Threonine Kinase	ATR	DNA damage
RAD50 Homolog	RAD50	DSBR; DNA recombination; Telomere maintenance
RAD52 Homolog	RAD52	DSBR; DNA recombination
MRE11 Homolog A	MRE11A	DSBR; DNA recombination; Telomere maintenance
Ubiquitin Conjugating Enzyme E2 I	UBE2I	DSBR; DNA recombination; Sumoylation
Ubiquitin Like Modifier Activating Enzyme 2	UBA2	Sumoylation
PMS1 Homolog 1	PMS1	MMR
PMS1 Homolog 2	PMS2	MMR; DNA damage
MutS Homolog 2	MSH2	MMR
MutS Homolog 3	MSH3	MMR
X-Ray Repair Cross Complementing 5	XRCC5	DSBR; Innate immune response
X-Ray Repair Cross Complementing 6	XRCC6	DSBR; Innate immune response
WRN RecQ Like Helicase	WRN	DNA repair; Homologous recombination

Abbreviations: PPI, protein-protein interaction; MMR, DNA mismatch repair; DSBR, Double-strand break repair.

Figure 6 Knockdown of EXO1 inhibited the migratory capacity of lung cancer cells. (A) The knockdown effect of EXO1 was detected by qRT-PCR. (B) The results of transwell migration assays among A549 and H1299 cells. (C) Knockdown of EXO1 significantly decreased migrated cell counts. *p<0.05; **p<0.01. p < 0.05 was considered to be significant.

Abbreviation: qRT-PCR, quantitative real-time PCR.