SLCO4A1 is a Prognosis-Associated Biomarker Involved in Neutrophil-Mediated Immunity in Thyroid Cancer

Figures & data

Figure 1 The acquisition process of the target gene. (A) Screening of the consistent genes that co-expressed with VEGFC. (B) Survival difference between the high and low expression of the 38 genes co-expressed with VEGFC. (C) The significant relationship between 11 co-expressed genes and PFS. (D) The differential expression analysis of SLCO4A1 and RNASEH1. (E) The correlation between SLCO4A1 expression and VEGFC expression. (F) The correlation between RNASEH1 expression and VEGFC expression. (G) The expression and survival analyses of SLCO4A1 in thyroid cancer. (H) The correlation between GDSC drug sensitivity and SLCO4A1 mRNA expression.

Figure 2 Overexpression of SLCO4A1 in thyroid cancer. (A) The gene expression level of SLCO4A1 in multiple types of tumor and normal tissues in TIMER. Red box indicated thyroid cancer. (B) SLCO4A1 gene expression in normal and thyroid cancer via UALCAN database. (C) The localization of SLCO4A1 protein in tumor cells and immunofluorescence staining in the A-431 cell line. (D and E) The protein expression of SLCO4A1 in thyroid cancer and normal tissues. *P <0.05, ***P <0.001.

Table 1 Correlation Between SLCO4A1 and Clinical Features in Thyroid Cancer

Variables	SLCO4A1 Expression		χ^2	P-value
	Low	High
Age (years)			1.022	0.312
< 55	152	128
≥ 50	56	59
M stage			0.275	0.600
M0	118	103
M1	3	5
N stage			0.125	0.724
N0	92	81
N1	97	92
T stage			3.424	0.331
T1	66	47
T2	63	69
T3	73	63
T4	5	7
Cancer stage			8.323	0.040
Stage 1	134	94
Stage 2	16	19
Stage 3	41	50
Stage 4	16	23
Race			0.009	0.995
Asian	21	20
African-American	9	9
Caucasian	132	126
Gender			0.065	0.799
Female	150	137
Male	58	50
Histological type			1.892	0.388
PTC-classical	153	129
PTC-follicular	39	38
PTC-tall cell	12	17
Primary tumor laterality			1.278	0.734
Bilateral	34	34
Isthmus	9	9
Left lobe	78	60
Right lobe	85	82

Abbreviation: PTC, papillary thyroid cancer.

Figure 3 SLCO4A1 expression at different stages in thyroid cancer. (A) UALCAN. (B) GEPIA.

Table 2 Cox Regression Analysis of the SLCO4A1 Expression and Progression-Free Survival in Thyroid Cancer Patients

Covariates	Univariate Analysis			Multivariate Analysis
	Hazard Ratio	95% CI	P-value	Hazard Ratio	95% CI	P-value
Age	1.026	1.005–1.047	0.015	1.003	0.970–1.038	0.841
Male	1.452	0.745–2.830	0.273	1.217	0.541–2.737	0.634
Stage 2	1.210	0.350–4.182	0.763	1.641	0.301–8.952	0.567
Stage 3	2.540	1.225–5.265	0.012	3.032	0.980–9.379	0.054
Stage 4	3.503	1.409–8.708	0.007	5.298	1.421–19.753	0.013
African-American	1.587	0.265–9.503	0.613	0.851	0.132–5.508	0.866
Caucasian	1.231	0.373–4.057	0.733	0.753	0.216–2.620	0.655
PTC- follicular	0.749	0.288–1.946	0.553	0.852	0.239–3.046	0.806
PTC- tall cell	2.528	1.040–6.142	0.041	1.971	0.722–5.379	0.185
Isthmus	0.350	0.044–2.774	0.320	0.616	0.074–5.109	0.653
Left lobe	0.549	0.233–1.294	0.171	0.704	0.271–1.826	0.470
Right lobe	0.629	0.275–1.438	0.271	0.737	0.291–1.868	0.520
SLCO4A1	1.007	1.004–1.009	<0.001	1.008	1.005–1.010	<0.001

Abbreviations: 95% CI, confidence interval; PTC, papillary thyroid cancer.

Table 3 Multivariate Analysis of the SLCO4A1 Expression and Progression-Free Survival in Thyroid Cancer Patients

Covariates	Hazard Ratio	95% CI-l	95% CI-u	P-value
Age	1.003	0.969	1.037	0.876
Male	1.192	0.521	2.724	0.677
Stage 2	1.647	0.300	9.031	0.566
Stage 3	3.146	1.007	9.826	0.049
Stage 4	5.450	1.453	20.435	0.012
African-American	0.802	0.122	5.283	0.819
Caucasian	0.706	0.202	2.465	0.585
PTC- follicular	0.843	0.231	3.080	0.796
PTC- tall cell	1.939	0.709	5.304	0.197
Isthmus	0.611	0.074	5.070	0.649
Left lobe	0.714	0.277	1.841	0.485
Right lobe	0.733	0.289	1.854	0.511
SLCO4A1	1.008	1.005	1.010	<0.001
VEGFC	1.000	0.999	1.001	0.941

Abbreviations: 95% CI-l, 95% confidence interval lower; 95% CI-u, 95% confidence interval upper; PTC, papillary thyroid cancer.

Table 4 The Correlation Between SLCO4A1 Expression and Prognosis in Thyroid Cancer Patients with Different Pathological Factors

Clinicopathological Characteristics	Overall Survival (n= 502)			Progression Free Survival (n= 353)
	Number	Hazard Ratio	P-value	Number	Hazard Ratio	P-value
Stage
1	281	/	0.14	234	5.79	0.0092
2	52	/	0.2	44	0.85	0.87
3	112	0.85	0.83	75	2.17	0.28
4	55	5.7	0.08	/	/	/
Gender
Female	367	0.69	0.57	269	3.03	0.024
Male	135	0.81	0.79	84	1.78	0.5
Race
Caucasian	332			227	2.4	0.064
Asian	51	/	1	43	0.84	0.9
African-American	27	/	0.21	12	/	/

Table 5 Multivariate Analysis of the SLCO4A1 Expression and Progression-Free Survival in Thyroid Cancer Patients at Stage 1 or Female Patients

Covariates	Stage 1			Female
	Hazard Ratio	95% CI-l-u	P-value	Hazard Ratio	95% CI-l-u	P-value
Age	0.944	0.865–1.029	0.191	0.961	0.910–1.016	0.159
Male	0.536	0.061–4.668	0.572	/	/	/
African-American	2.781	0.000-Inf	0.999	0.621	0.057–6.759	0.696
Caucasian	336,973.591	0.000-Inf	0.976	0.490	0.133–1.805	0.284
SLCO4A1	1.009	1.005–1.014	<0.001	1.008	1.005–1.011	<0.001
VEGFC	1.000	0.998–1.002	0.995	1.000	0.999–1.002	0.616
Stage 2	/	/	/	4.420	0.671–29.141	0.122
Stage 3	/	/	/	7.209	1.549–33.545	0.012
Stage 4	/	/	/	12.293	1.723–87.681	0.012
PTC- follicular	0.874	0.100–7.642	0.903	0.325	0.039–2.680	0.296
PTC- tall cell	5.281	0.518–53.890	0.160	1.492	0.445–5.001	0.517
Isthmus	0.000	0.000-Inf	0.988	0.732	0.078–6.896	0.785
Left lobe	0.927	0.141–6.089	0.937	0.818	0.250–2.674	0.740
Right lobe	0.778	0.140–4.311	0.774	0.613	0.190–1.979	0.414

Abbreviations: 95% CI-l, 95% confidence interval lower; 95% CI-u, 95% confidence interval upper; PTC, papillary thyroid cancer.

Figure 4 The high SLCO4A1 expression predicted poor PFS in thyroid cancer patients at stage 1 and female patients. (A) Stage 1. (B) Female.

Figure 5 Receiver operating characteristic curves. (A) Overall survival. (B) Progression-free survival.

Figure 6 Screening and enrichment analysis of SLCO4A1 and its co-expressed genes. (A) The volcano plot of the genes co-expressed with SLCO4A1. (B) The 50 positively correlated genes. (C) The 50 negatively correlated genes. (D) Gene ontology enrichment analysis. (E) KEGG pathway enrichment analysis.

Figure 7 The correlation between SLCO4A1 expression and neutrophil infiltration as well as its gene markers. The correlation between SLCO4A1 and neutrophil through (A) TIMER, and (B) TISIDB. The correlation between SLCO4A1 and neutrophil gene marker CEACAM8 through (C) TIMER, and (E) GEPIA. The correlation between SLCO4A1 and neutrophil gene marker FUT4 through (D) TIMER, and (F) GEPIA.

Figure 8 The association of SLCO4A1 with immunoregulators in thyroid cancer. (A) CD274. (B) PDCD1LG2. (C) IL2RA.