Solute carrier family 2 members 1 and 2 as prognostic biomarkers in hepatocellular carcinoma associated with immune infiltration

Figure 1 Solute carrier family 2 member 1 expression was increased whereas solute carrier family 2 member 2 expression was decreased in liver cancer. A: The reads per kilobase million (RPKM) of solute carrier family 2 in normal liver tissues from 95 human individuals by National Center for Biotechnology Information's gene database. The RPKM of solute carrier family 2 member 2 (SLC2A2) was 193.619, while solute carrier family 2 member 1 (SLC2A1) was almost undetectable; B: The fragments per kilobase million (FPKM) of solute carrier family 2 in HepG2 cells by RNA sequencing. The FPKM of SLC2A1 was 314.336 while SLC2A2 was 2.68857 in HepG2 cells; C: The FPKM of solute carrier family 2 in HepG2215 cells by RNA sequencing. The FPKM of SLC2A1 was 412.048 while SLC2A2 was 12.0905 in HepG2215 cells; D: The messenger RNA expression level of SLC2A1 was increased while that of SLC2A2 was decreased in hepatocellular carcinoma (HCC) in the cohort [gene expression series 121248 (GSE121248)]; E: The messenger RNA expression level of SLC2A1 was increased while that of SLC2A2 was decreased in HCC compared with normal tissues in The Cancer Genome Atlas (TCGA); F: Immunofluorescence analysis revealed that SLC2A1 protein expression was expressed and SLC2A2 protein expression was weakly expressed in HepG2 cells in the Human Protein Atlas; G: Immunohistochemical analysis showed the expression level of SLC2A1 was increased while SLC2A2 was decreased in liver cancer compared with normal tissues in the Human Protein Atlas. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 2 Solute carrier family 2 member 1 and solute carrier family 2 member 2 messenger RNA expression levels in different human cancers were changed by using tumor immune estimation resource and ONCOMINE databases. A: The messenger RNA expression levels of solute carrier family 2 member 1 (SLC2A1) and solute carrier family 2 member 2 (SLC2A2) in different types of human cancers by the Tumor Immune Estimation Resource database; B: The messenger RNA expression levels of SLC2A1 and SLC2A2 in different human cancers by ONCOMINE database. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. TPM: Transcripts per million; ACC: Adrenocortical carcinoma; BLCA: Bladder urothelial carcinoma; BRCA: Breast invasive carcinoma; CESC: Cervical squamous cell carcinoma and endometrial adenocarcinoma; CHOL: Cholangiocarcinoma; COAD: Colon adenocarcinoma; ESCA: Esophageal carcinoma; GBM: Glioblastoma multiforme; HNSC: Head and neck cancer; KICH: Kidney chromophobe; KIRC: Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; LAML: Acute Myeloid Leukemia; LGG: Brain Lower Grade Glioma; LIHC: Liver hepatocellular carcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; MESO: Mesothelioma; OV: Ovarian serous cystadenocarcinoma; PAAD: Pancreatic adenocarcinoma; PCPG: Pheochromocytoma and paraganglioma; PRAD: Prostate adenocarcinoma; READ: Rectum adenocarcinoma; SARC: Sarcoma; SKCM: Skin Cutaneous Melanoma; STAD stomach adenocarcinoma; TGCT: Testicular Germ Cell Tumors; THCA: Thyroid carcinoma; THYM: Thymoma; UCEC: Uterine corpus endometrial carcinoma; UCS: Uterine carcinosarcoma; UVM: Uveal melanoma.

Figure 3 Solute carrier family 2 member 1 and solute carrier family 2 member 2 transcription levels in hepatocellular carcinoma patients were correlated with tumor volume, grade, and stage. A: Solute carrier family 2 member 1 (SLC2A1) level gradually increased whereas solute carrier family 2 member 2 (SLC2A2) level gradually decreased with the process of primary tumor volume enlargement in The Cancer Genome Atlas (TCGA) data; B: SLC2A1 level gradually increased, whereas SLC2A2 level gradually decreased with the process of hepatocellular carcinoma (HCC) grade in TCGA data; C: SLC2A1 level gradually increased whereas SLC2A2 level gradually decreased with the process of HCC grade in the UALCAN database; D: SLC2A1 level gradually increased, whereas SLC2A2 level gradually decreased with the process of HCC stage in UALCAN database. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 4 Solute carrier family 2 member 1 and solute carrier family 2 member 2 messenger RNA levels were correlated with survival rates of hepatocellular carcinoma patients in The Cancer Genome Atlas data and the Kaplan-Meier Plotter database. A: In The Cancer Genome Atlas (TCGA) database, the top graph represents the scatter graph of solute carrier family 2 member 1 (SLC2A1) expression from low to high, where the blue represented the low expression group and the red represented the high expression group. The middle graph represents the scatter graph of the survival time and survival state corresponding to the SLC2A1 expression level of different samples. The bottom graph is a heat map of the expression of SLC2A1 in diverse samples; B: According to the expression level of SLC2A1, the patients were divided into the high expression group and the low expression group, and the results suggested that the low expression group had better overall survival (OS); C: Area under the curve (AUC) of SLC2A1 in 1, 3, and 5 years. The higher the value was, the stronger the predictive ability of the gene was; D: In TCGA database, the top graph represents the scatter graph of solute carrier family 2 member 2 (SLC2A2) expression from low to high, where the blue represents the low expression group and the red represents the high expression group. The middle graph represents the scatter graph of the survival time and survival state corresponding to the SLC2A2 expression level of different samples. The bottom graph is a heat map of the expression of SLC2A2 in diverse samples; E: According to the expression level of SLC2A2, the patients were divided into the high expression group and the low expression group and the results suggested that the low expression group had poor OS; F: AUC of SLC2A2 in 1, 3, and 5 years. The higher the value was, the stronger the prognostic ability of the gene; G: Hepatocellular carcinoma (HCC) patients with high expression of SLC2A1 had poorer OS [hazard ratio (HR): 2.25, P = 3.7e-06] and relapse-free survival (RFS) (HR: 1.4, P = 0.075); H: HCC patients with high expression of SLC2A2 had better OS (HR: 2.25, P = 3.7e-06) and RFS (HR: 0.55, P = 6.6e-04).

Figure 5 Solute carrier family 2 member 1 and solute carrier family 2 member 2 showed negative correlations in hepatocellular carcinoma patients in multiple databases. A: solute carrier family 2 member 1 (SLC2A1) was negatively correlated with solute carrier family 2 member 2 (SLC2A2) in the cohort gene expression series 121248 (r = -0.4756, P < 0.0001); B: SLC2A1 was negatively correlated with SLC2A2 in The Cancer Genome Atlas data (r = -0.28, P = 6.41e-08); C: SLC2A1 was negatively correlated with SLC2A2 in the Tumor Immune Estimation Resource database (r = -0.252, P = 8.4e-07); D: SLC2A1 was negatively correlated with SLC2A2 in the gene expression profiling interactive analysis database (r = -0.19, P = 1.3e-04); TPM: Transcripts per million.

Figure 6 The String database was used to predict the effects of solute carrier family 2 member 1, solute carrier family 2 member 2, and frequently altered adjacent genes expression changes on functions and pathways. A: The protein-protein interaction networks of solute carrier family 2 member 1 (SLC2A1), solute carrier family 2 member 2 (SLC2A2), and their neighbor genes in the String database were constructed, which contained 22 nodes and 107 edges. In this networks diagram, genes tumor protein p53, ATM serine/threonine kinase, MDM2 proto-oncogene, protein kinase-B, hypoxia inducible factor 1 subunit alpha, binding protein p300, insulin, glucokinase, and hexokinase 2 were included; B: SLC2A1, SLC2A2, and their neighbors were analyzed by Kyoto Encyclopedia of Genes and Genomes, and the top 20 genes were ranked by P value from small to large in the String database; C: SLC2A1, SLC2A2, and their neighbors were analyzed by biological process (BP), and the top 20 genes were ranked by P value from small to large in the String database; D: SLC2A1, SLC2A2, and their neighbors were analyzed by biological process (MF), and the top 20 genes were ranked by P value from small to large in the String database; E: SLC2A1, SLC2A2, and their neighbors were analyzed by cellular component (CC), and the top 20 genes were ranked by P value from small to large in the String database. HIF1AN: Hypoxia inducible factor 1 subunit alpha inhibitor; GSK3B: Glycogen synthase kinase 3 beta; GIPC1: GIPC PDZ domain containing family member 1; CREBBP: CREB binding protein; BCL2L1: BCL2 Like 1; VHL: von Hippel-Lindau tumor suppressor; SMUG1: Single-strand-selective monofunctional uracil-DNA glycosylase 1; SLC5A1: Solute carrier family 5 member 1; NOS3: Nitric oxide synthase 3; INSR: Insulin receptor; HNF1A: HNF1 homeobox A.

Figure 7 The landscape of infiltrating immune cells in hepatocellular carcinoma was different from that in normal liver tissues and expressions of solute carrier family 2 member 1 and solute carrier family 2 member 2 correlated with immune infiltration level in hepatocellular carcinoma. A and B: The pattern of immune cells by the signature gene expression profile in hepatocellular carcinoma compared with normal samples with the Cell Type Identification by Estimating Relative RNA Transcript subsets method. In comparison to normal tissues, the proportions of B cell memory, regulatory T, T cell gamma delta, monocyte, macrophage M0, macrophage M2, myeloid dendritic cell resting, mast cell activated, mast cell resting, and neutrophil had changed; C: Correlation analysis between solute carrier family 2 member 1 (SLC2A1) transcription level and immune cell infiltration level. The immune cells included B cells (partial.cor (r) = 0.291), CD8⁺ T cells (r = 0.296), CD4⁺ T cells (r = 0.238), macrophages (r = 0.425), neutrophils (r = 0.424), and dendritic cells (r = 0.368); D: Correlation analysis between solute carrier family 2 member 1 (SLC2A2) transcription level and immune cell infiltration level. The immune cells included B cells (r = -0.188), CD8⁺ T cells (r = -0.188), macrophages (r = -0.148), neutrophils (r = -0.115) and dendritic cells (r = -0.17). ^aP < 0.05; ^cP < 0.001.

Figure 8 Solute carrier family 2 member 1 and solute carrier family 2 member 2 expressions correlated with immune marker genes in hepatocellular carcinoma by using tumor immune estimation resource and gene expression profiling interactive analysis databases. A: Solute carrier family 2 member 1 (SLC2A1) expression correlated with macrophage polarization in hepatocellular carcinoma (HCC). Markers included CD86 and CD115 of monocytes; C-C motif chemokine ligand 2, CD68, and interleukin (IL) 10 of tumor-associated macrophage; nitric oxide synthase 2, interferon regulatory factor 5, and COX2 cytochrome c oxidase subunit II of M1 macrophages; and CD163, V-set and immunoglobulin domain containing 4, and membrane spanning 4-domains A4A of M2 macrophages; B: Solute carrier family 2 member 2 (SLC2A2) expression correlated with macrophage polarization in HCC. Markers included CD86 and CD115 of monocytes; C-C motif chemokine ligand 2, CD68, and IL10 of tumor-associated macrophage; nitric oxide synthase 2, interferon regulatory factor 5, and COX2 cytochrome c oxidase subunit II of M1 macrophages; and CD163, V-set and immunoglobulin domain containing 4, and membrane spanning 4-domains A4A of M2 macrophages; C: SLC2A1 expression was correlated with functional T cells in HCC. Markers included BCL6 transcription repressor and IL21 of follicular helper T; C-C motif chemokine receptor 8, signal transducer and activator of transcription (STAT) 5B, and transforming growth factor-beta markers of T cell regulatory; STAT1, interferon gamma, and tumor necrosis factor of T helper (Th)1; and GATA binding protein 3, STAT6, and STAT5A of Th2; D: SLC2A2 expression was correlated with functional T cells in HCC. Markers included BCL6 transcription repressor and IL21 of follicular helper T; C-C motif chemokine receptor 8,, STAT5B, and transforming growth factor-beta markers of T cell regulatory; STAT1, interferon gamma, and tumor necrosis factor alpha of Th1 and GATA binding protein 3, STAT6 and STAT5A of Th2; E: SLC2A1 expression correlated with T cell exhaustion in HCC. Markers included programmed cell death 1, cytotoxic T-lymphocyte associated protein 4, lymphocyte activating 3, hepatitis A virus cellular receptor 2, and granzyme B; F: SLC2A2 expression correlated with T cell exhaustion in HCC. Markers included programmed cell death 1, cytotoxic T-lymphocyte associated protein 4, lymphocyte activating 3, hepatitis A virus cellular receptor 2, and granzyme B. TAM: Tumor-associated macrophage; CCL2: C-C motif chemokine ligand 2; IL10: interleukin 10; NOS2: nitric oxide synthase 2; IRF5: interferon regulatory factor 5; PTGS2: COX2 cytochrome c oxidase subunit II;VSIG4: V-set and immunoglobulin domain containing 4; MS4A4A: membrane spanning 4-domains A4A; Tfh: Follicular helper T; BCL6: BCL6 transcription repressor; IL21: Interleukin 21; Treg: T cell regulatory; CCR8: C-C motif chemokine receptor 8; STAT5B: signal transducer and activator of transcription 5B; TGFB1: transforming growth factor-beta; Th: T helper; STAT1: signal transducer and activator of transcription 1; IFNG: interferon gamma; TNF: tumor necrosis factor; GATA3: GATA binding protein 3; STAT6: signal transducer and activator of transcription 6; STAT5A: signal transducer and activator of transcription 5A; PDCD1: programmed cell death 1; CTLA4: cytotoxic T-lymphocyte associated protein 4; LAG3: lymphocyte activating 3; HAVCR2: hepatitis A virus cellular receptor 2; GZMB: granzyme B.

Figure 9 Solute carrier family 2 member 2 methylation correlated with immune cells in hepatocellular carcinoma by using the tumor and immune system interaction database. A: Heat map of the correlation between solute carrier family 2 member 2 (SLC2A2) transcription level and the level of immune cell infiltration; B: Scatter plot of the correlation between SLC2A2 transcription level and the level of immune cell infiltration.Act: activated; Tcm: central memory T; Tem: effector memory T; Tfh: Follicular helper T; Tgd: gamma-delta T cells; Th: T helper; Imm: immune; Mem: memory; NK: natural killer; MDSC: myeloid-derived suppressor; NKT: natural killer T; pDC: plasmacytoid dendritic cells; iDC: interstitial dendritic cells.