High Expression of SLC41A3 Correlates with Poor Prognosis in Hepatocellular Carcinoma

Figures & data

Figure 1 Expression of SLC41A3 mRNA in HCC and normal tissues. (A) The expression of SLC41A3 mRNA in various cancers based on the TCGA database (UALCAN). (B–E) Box plot showing SLC41A3 mRNA levels in the Roessler Liver, Roessler Liver 2, Wurmbach Liver, and Mas Liver datasets, respectively (Oncomine). (F) Comparison of SLC41A3 mRNA level across 4 analyses showed a significant difference between HCC and normal tissues.

Figure 2 SLC41A3 transcription in subgroups of HCC patients stratified by grade, stage, and other criteria (UALCAN). (A) Boxplot showing relative expression of SLC41A3 in HCC and normal tissue samples. (B–D) Association between SLC41A3 expression and histological grade of HCC (B), TNM stage of HCC (C), and nodal metastasis status of HCC (D). Data are mean ± SE. *P<0.05; **P<0.01; ***P<0. 001.

Table 1 Expression of SLC41A3 in Hepatocellular Carcinoma Tissues (n=323)

Clinical Parameters			Patients	SLC41A3 Expression		OR	χ^2	P-value
				Low (%)	High (%)
Age (years)						0.92	0.10	0.75
	<55		125	34 (27.20)	91 (72.80)
	≥55		198	57 (28.79)	141(72.21)
Gender						0.76	0.79	0.37
	Male		262	71 (27.10)	191 (72.90)
	Female		61	20 (32.79)	41 (67.21)
Tumor size						1.32	1.22	0.27
	<5cm		169	52(30.78)	117 (69.22)
	≥5cm		147	37 (25.17)	110 (74.83)
Tumor number						1.44	1.16	0.28
	Single		265	78 (29.43)	187 (70.57)
	Multiple		58	13 (22.41)	45 (77.59)
Edmondson grade						1.88	5.39	0.02
	I+II		202	65 (32.18)	137 (67.82)
	III		119	24 (20.17)	95 (79.83)
Metastasis						3.25	3.92	0.04
	Negative		292	87 (29.79)	205 (70.21)
	Positive		26	3 (11.54)	23 (88.46)
Microvascular invasion						2.04	5.97	0.01
	Absence		121	42 (34.71)	79 (65.29)
	Presence		121	25 (20.66)	96 (79.34)
AFP (μg/l)						1.74	4.10	0.04
	<50		140	49 (35.00)	91 (65.00)
	≥50		123	29 (23.58)	94 (76.42)
Cirrhosis						1.23	0.68	0.41
		Negtive	106	33 (31.13)	73 (68.87)
		Positive	217	58 (26.73)	159 (73.27)

Figure 3 Immunohistochemical staining of SLC41A3 in HCC and normal tissues. (A) High level of SLC41A3 expression in HCC. (B) Low level of SLC41A3 expression in HCC. (C) Immunohistochemical staining of SLC41A3 in normal hepatic tissue. Original magnification: ×40 (left column) and ×200 (right column).

Table 2 Univariate and Multivariate Cox Regression Survival Analysis of Clinicopathological Parameters and SLC41A3 Expression in Hepatocellular Carcinoma Patients

Parameters	Univariate Analysis			Multivariate Analysis
	HR	95% CI	P	HR	95% CI	P
Age	0.65	0.41–1.04	0.07	NA
Gender	1.58	0.92–2.69	0.09	NA
Tumor size	1.95	1.22–3.13	<0.01	1.13	0.54–2.38	0.75
Tumour number	1.24	0.68–2.26	0.49	NA
Edmondson Grade	2.78	1.74–4.46	<0.01	2.55	1.21–5.36	0.01
Metastasis	4.82	2.55–9.11	<0.01	3.02	1.11–8.22	0.03
Microvascular invasion	2.11	1.25–3.56	<0.01	1.36	0.66–2.81	0.41
HBs antigen	1.17	0.65–2.10	0.60	NA
Cirrhosis	1.15	0.70–1.91	0.58	NA
AFP level	2.51	1.40–4.52	<0.01	1.66	0.81–3.41	0.17
SLC41A3 expression	2.56	1.43–4.60	<0.01	1.58	0.70–3.54	0.27

Abbreviations: HBs antigen, hepatitis B surface antigen; AFP, alpha fetoprotein; HR, hazard ratio; CI, confidential interval.

Figure 4 Survival analysis in HCC patients with high or low SLC41A3 expression. (A) High expression of SLC41A3 is associated with poor survival (Kaplan–Meier Plotter database). (B) Kaplan–Meier survival curves of HCC patients recruited in the present study with high or low SLC41A3 expression show that the higher was the expression of SLC41A3 in cancer tissues, the lower was the survival rate.

Figure 5 Genes co-expressed with SLC41A3 in HCC (LinkedOmics). (A) Pearson’s test was used to analyze correlations between SLC41A3 and genes differentially expressed in HCC. (B and C) Heat maps showing the top 50 genes positively and negatively correlated with SLC41A3 in HCC. Red indicates positively correlated genes, and green indicates negatively correlated genes. (D–F) The positive correlation between SLC41A3 and the top three genes (ELOVL1, HDAC11, and NEU1). (G–I) The negative correlation between SLC41A3 and the top three genes (CYP4V2, SOCS2, and USP38).

Figure 6 Significantly enriched GO annotations and KEGG pathways of SLC41A3 in HCC (LinkedOmics). The significantly enriched GO annotations and KEGG pathways of genes co-expressed with SLC41A3 in HCC were analyzed using gene set enrichment analysis. (A) Cellular components. (B) Biological processes. (C) Molecular functions. (D) KEGG pathway analysis. The x-axis represents the normalized enrichment score, and the y-axis represents the term of GO.

Table 3 Gene Sets Enriched in High SLC41A3 Expression Phenotype in HCC

Name	ES	NES	NOM P-val	FDR q-val
KEGG_CELL_CYCLE	0.73	1.97	0.000	0.003
KEGG_APOPTOSIS	0.59	1.87	0.002	0.008
KEGG_mTOR_SIGNALING_PATHWAY	0.61	1.83	0.002	0.011
KEGG_PATHWAYS_IN_CANCER	0.59	1.98	0.000	0.003
KEGG_TGF_BETA_SIGNALING_PATNWAY	0.59	1.81	0.002	0.012
KEGG_WNT_SIGNALING_PATHWAY	0.61	1.98	0.000	0.003

Notes: Gene sets with NOM p-value<0.05 and FDR q-value<0.05 are considered as significant.

Abbreviations: ES, enrichment score; NES, normalized enrichment score; NOM, nominal; FDR, false discovery rate.

Figure 7 GSEA reveals potential signaling pathways of SLC41A3. The analysis showed that the cell cycle (A), apoptosis (B), mTOR signaling pathway (C), pathways in cancer (D), TGF-β signaling pathway (E), and WNT signaling pathway (F) were enriched in the SLC41A3 high-expression group. The top panels indicate the enrichment scores for each gene, while the bottom panels show the ranking metrics of each gene. Y-axis: ranking metric values; X-axis: ranks for all genes.

Abbreviation: NES, normalized enrichment score.

Figure 8 Protein–protein interaction network of SLC41A3. Interactions between SLC41A3 and other genes were obtained from the web portals STRING (A) and GeneMANIA (B).