SLC1A5 overexpression accelerates progression of hepatocellular carcinoma by promoting M2 polarization of macrophages

doi:10.12122/j.issn.1673-4254.2025.02.08

Abstract

Abstract:

Objective To investigate the clinical significance of SLC1A5 overexpression in pan-cancer and its mechanism for promoting hepatocellular carcinoma (HCC) progression. Methods We analyzed the correlation of SLC1A5 expression with clinical stage, lymph node metastasis and prognosis in pan-cancer using TCGA and ICGC datasets and explored its association with immune cell infiltration using EPIC, CIBERSORT, and TIMER algorithms. In HCC cell lines, the effects of lentivirus-mediated SLC1A5 overexpression or RNA interference on cell proliferation were examined using CCK-8 assay, and the growth of HCC cell xenografts overexpressing SLC1A5 was observed in nude mice. The effects of SLC1A5 overexpression or silencing in HCC cells on macrophage polarization were evaluated in a cell co-culture system. Results SLC1A5 was mainly localized on cell membrane and was highly expressed in most cancers in association with clinical stage, lymph node metastasis and poor prognosis. SLC1A5 expression was positively correlated with immunity score in 13 cancer types, especially in low-grade glioma (LGG), LIHC and thyroid cancer. SLC1A5 was positively correlated with macrophage infiltration level in LGG and LIHC but negatively correlated with macrophage infiltration in 5 cancers including lung squamous carcinoma, pancreatic carcinoma, and gastric carcinoma. Patients with SLC1A5 overexpression and high level of M2 macrophage infiltration had the worst survival outcomes. SLC1A5 was correlated with immunosuppression-related genes, cytokines, and cytokine receptors, which was the most obvious in LGG and LIHC. SLC1A5 was highly expressed in different HCC cell lines, and its overexpression promoted HCC cell proliferation both in vitro and in nude mice. In the cell co-culture experiment, SLC1A5 was positively correlated with the molecular markers of M2 polarization of macrophages, and its overexpression strongly promoted M2 polarization of the macrophages and inhibited T cell secretion of IFN-γ. Conclusion SLC1A5 expression level is correlated with clinical stage, lymph node metastasis, prognosis, and immune cell infiltration in most cancers, and its overexpression promotes HCC progression by inhibiting T-cell function via promoting M2 polarization of macrophages.

Key words: pan-cancer, SLC1A5, M2 macrophages, hepatocellular carcinoma, proliferation

Jinhua ZOU, Hui WANG, Dongyan ZHANG. SLC1A5 overexpression accelerates progression of hepatocellular carcinoma by promoting M2 polarization of macrophages[J]. Journal of Southern Medical University, 2025, 45(2): 269-284.

Figures/Tables 12

Tab.1 siRNA sequences for SLC1A5

Products name	Target sequence
SLC1A5(siRNA# 1)	5'-GTACCGTCCTCAATGTAGA-3'
SLC1A5(siRNA# 2)	5'- GAAGCACAGAGCCTGAGTT-3'

Tab.2 PCR primer sequences

Primer

Sequence

SLC1A5

Sense

Antisense

5'-GAGGCTTTCTCTGGCTGGTAA-3'

5'-GTCTTGGACACTGAGGGCTG-3'

TGFB1

IL-10

CSF1R

CXCL1

Sense

Antisense

Sense

Antisense

Sense

Antisense

Sense

Antisense

5'-CAAGTGGACATCAACGGGTTC-3'

5'-GCAGCAGTTCTTCTCCGTGG-3'

5'-GAGGAGGTGATGCCCCAAGC-3'

5'-GGGAAGAAATCGATGACAGC-3'

5'-CCTCGCTTCCAAGAATTGCA-3'

5'-CCCAATCTTGGCCACATGA-3'

5'-CTCTTCCGCTCCTCTCACAG-3'

5'-CACGGACGCTCCTGCTGC-3'

Fig1 Differential expressions of SLC1A5 across different cancer types. A: SLC1A5 expression in normal tissues. B: SLC1A5 expression in tumor cell lines. C: Expression status of SLC1A5 gene in paired tumor and non-tumor tissues across 22 cancer types from TCGA. D: Analysis of SLC1A5 expression in 33 cancer types with their corresponding normal tissues in GTEx dataset as controls. *P<0.05, **P<0.01, *** P<0.001.

Fig.2 SLC1A5 is highly expressed in various cancers. A: Expression and distribution of SLC1A5 in different cancer cell lines (Scale bar=10 μm); B-F: Comparison of SLC1A5 gene expression between normal and tumor tissues from TCGA databases (left), SLC1A5 protein expression between normal and tumor tissues from CPTAC (middle), and immunohistochemistry images of normal and tumor tissues from HPA (right, Scale bar=100 μm). SLC1A5 expression at both the mRNA and protein levels was significantly higher in colorectal cancer (B), lung cancer (C), and endometrial cancer (D). There was no significant difference in SLC1A5 expression between tumor and non-tumor in breast cancer (E). SLC1A5 gene expression was slightly upregulated in renal cancer, but was significantly downregulated in the tumor tissues at the protein level (F). *P<0.05, ***P<0.001.

Fig.3 SLC1A5 is highly expressed in various cancer types. Comparison of SLC1A5 gene expression between normal and tumor tissues from TCGA databases (left), SLC1A5 protein expression between normal and tumor tissues from CPTAC (middle), and immunohistochemistry images of normal and tumor tissues from HPA (right, Scale bar=100 μm). ***P<0.001.

Fig.4 Expression levels of SLC1A5 gene in cancers in realtion to pathological stages and status of lymphatic metastasis. A: Expression levels of SLC1A5 mRNA in different pathological stages of HNSC, KIRC, KIRP, LIHC, PAAD, TGCT, and THCA. B: Expression levels of SLC1A5 mRNA analyzed in relation to lymphatic metastasis in HNSC, KIRC, LIHC, and THCA. *P<0.05, **P<0.01, ***P<0.001.

Fig.5 Correlation between SLC1A5 expression and survival outcomes of different cancers. A: GEPIA2 tool was used for analysis of overall survival (A) and disease-survival (B) for 33 cancers in TCGA in relation to SLC1A5 expression. The survival map and survival curves with statistical significance are shown.

Fig.6 Correlation of SLC1A5 expression levels with tumor microenvironment and levels of tumor immune cell infiltration. A: Three tumors with the highest correlation coefficients between SLC1A5 expression and the tumor microenvironment. B: Correlation coefficients between SLC1A5 expression and tumor infiltration level of different immune cells in 33 cancer types. *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.

Fig.7 Co-expression of SLC1A5 expression and immune-related genes. Correlations of SLC1A5 expression with immunosuppressive genes (A), immune activation genes (B), chemokines receptor (C), and chemokines (D) are presented.

Fig.8 Correlation between SLC1A5 expression and different subtypes of macrophage. A: Relationship between SLC1A5 expression and different subtypes of macrophage. B: Three tumors with the highest correlation coefficients between SLC1A5 expression and M2 macrophages. C: Survival map illustrating the correlation between different subtypes of macrophage and overall survival in different cancers. D: Kaplan-Meier survival analysis of LGG (left) and LIHC (right) patients with different SLC1A5 expression levels stratified by levels of infiltrating M2 macrophages.

Fig.9 Overexpression of SLC1A5 promotes proliferation of HCC cells. A: Expression of SLC1A5 in HCC cell lines detected by Western blotting. LE: Long exposure; SE: Short exposure. B: Verification of SLC1A5 knockout and overexpression efficiency in different HCC cell lines using Western blotting. C: Effect of RNA interference of SLC1A5 on HCC cell proliferation assessed using CCK-8 assay. D: Effect of SLC1A5 overexpression on HCC cell proliferation assessed using by CCK-8 assay. E: Overexpression efficiency of SLC1A5 in HCC-LM3 cells examined by Western blotting (upper). Examination of subcutaneous tumors in nude mice (bottom). F, G: Proliferation curve of subcutaneous tumor (F) and tumor weight (G) in nude mice. H, I: Expressions of SLC1A5 and Ki-67 in subcutaneous tumors detected by immunohistochemistry. *P<0.05, **P<0.01, ***P<0.001.

Fig.10 SLC1A5 promotes M2 polarization of macrophages and induces CD8+ T cell dysfunction. A: Correlation analysis between SLC1A5 expression and molecular markers of M2 macrophages. B: M0 cells co-cultured with HCC cells transfected with SLC1A5-specific siRNA or negative control siRNA and induced to polarize into M2 phenotype using IL-4 (20 ng/mL). Interference efficiency of SLC1A5 and expressions of M2 markers (IL-10, TGFB1, CSF1R and CXCL-1) detected using qRT-PCR. C: M0 cells co-cultured with SLC1A5-overpressing HCC-LM3 cells and induced to polarize into M2 phenotype using IL-4 (20 ng/mL). The overpression efficiency of SLC1A5 and the expression of M2 markers (IL-10, TGFB1, CSF1R and CXCL-1) were detected using qRT-PCR. D, E: M0 cells co-cultured with HCC cells with SLC1A5 knockdown (D) and overexpression (E) and induced into the M2 phenotype using IL-4 (20 ng/mL). The culture medium was used to treat activated Jurkat T cells for 48 h, and IFN-γ secretion was detected by ELISA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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