Pan-cancer analysis of STX7 expression and genomic alterations

A comprehensive analysis of STX7 mRNA expression across cancer types was performed using data from the TCGA and GTEx databases. This analysis revealed significant alterations in STX7 expression across 23 cancer types (Fig. 1A). Additionally, we examined potential genomic alterations in STX7 across specific cancers. The findings showed that STX7 amplification was most common in SARC, while deep deletions were frequent in UVM and DLBC (Fig. 1B). STX7 expression was positively correlated with tumor mutational burden (TMB) in ESCA and THYM (Fig. 1C) and with microsatellite instability (MSI) in COAD, READ, and UCEC (Fig. 1D). These results suggest a significant association between STX7 expression and genomic instability across various cancer types.

Fig. 1

Pan-cancer analysis of STX7 expression, genomic alterations, and prognostic significance. (A)Integrated analysis of STX7 expression in tumor and healthy tissue samples was conducted using the TCGA and GTEx datasets. (B) Pan-cancer investigation of genomic alterations in STX7 was performed using data from the TCGA database, examining mutations, amplifications, and deep deletions. Radar plots were utilized to represent the correlation between STX7 and tumor mutational burden (TMB) (C) as well as microsatellite instability (MSI) (D). (E) A heatmap was generated to show the association between STX7 expression and overall survival (OS), disease-specific survival (DSS), disease-free survival (DFS), and progression-free survival (PFS)

Diagnostic and prognostic value of STX7 in multiple cancers

The ROC curve indicates that STX7 could serve as a potential diagnostic biomarker for certain cancers (Figure S1). To further investigate its prognostic value, we analyzed 33 cancer types from the TCGA database and examined the correlation between STX7 expression and various survival outcomes, including OS, DSS, PFS, and DFS. Univariate Cox regression analysis showed that high STX7 expression was significantly associated with poor OS in BRCA, CESC, LIHC, LUSC, and OV, while acting as a protective factor in KIRC. For DSS, STX7 was a risk factor for BRCA, CESC, HNSC, LUSC, and UCEC, and a protective factor in KIRC. For DFS, STX7 was a risk factor in CESC and HNSC, but a protective factor in LGG and UCS. For PFS, STX7 was a risk factor for CESC, HNSC, LUSC, and LIHC, and a protective factor in KIRC, THCA, LGG, and UCS. Kaplan-Meier survival analysis confirmed these findings (Fig. 1E). In summary, elevated STX7 expression was generally associated with poorer prognosis in patients with BRCA, CESC, LIHC, LUSC, HNSC, and UCEC.

GSEA of STX7 in pan-cancer

To identify cancer-related markers linked to STX7, we conducted GSEA on DEGs between the low and high STX7 subgroups in each cancer type. STX7 expression was significantly associated with immune-related pathways, including TNF-NFκB signaling, IFN-α and IFN-γ responses, inflammatory response, and allograft rejection pathways, particularly in BLCA, BRCA, COAD, HNSC, KICH, LAML, LUAD, LUSC, and SARC. These results imply a potential link between STX7 expression and immune activation within the tumor microenvironment (TME). Moreover, in the high STX7 subgroup of BLCA, BRCA, COAD, HNSC, LIHC, LUAD, LUSC, MESO, OV, PAAD, PRAD, READ, SARC, STAD, THYM, and UCEC, markers of epithelial-mesenchymal transition (EMT) were significantly enriched. suggesting that STX7 may contribute to cancer progression via EMT. Furthermore, the KRAS signaling pathway, IL6/JAK/STAT3 pathway, and IL-2/STAT5 pathway were strongly linked to STX7 expression in cancer. These findings underscore the potential of STX7 as a key player in immune activation and cancer progression, providing new avenues for further investigation (Fig. 2).

Fig. 2

Hallmark gene set enrichment analysis (GSEA) of STX7 in pan-Cancer. This analysis demonstrates the enrichment of hallmark gene sets associated with STX7 across different cancer types. The circle size reflects the false discovery rate (FDR) for each enriched term in the respective cancers, and the color represents the normalized enrichment score (NES) for each term

Role of STX7 in cancer immune infiltration

To investigate the immunological role of STX7 within the cancer microenvironment, we calculated the ESTIMATE score for STX7 across a range of cancer types. As shown in Fig. 3A, a positive correlation was observed between STX7 and the ESTIMATEScore, StromalScore, and ImmuneScore across several cancer types, including those from TCGA, such as BLCA, BRCA, COAD, HNSC, LAML, LIHC, LUAD, LUSC, OV, PAAD, and PRAD. However, in certain cancers, such as SKCM and THCA, STX7 exhibited a negative correlation with these scores. Additionally, cancers exhibiting a positive correlation between STX7 and ImmuneScore also showed a positive association with most immune checkpoint genes (Fig. 3B). Notably, genes such as HAVCR2, PDCD1LG2, CD274, and TIGIT exhibited the strongest positive correlations with STX7 across more than 20 cancer types, suggesting a role for STX7 in immune checkpoint-mediated regulation.

Fig. 3

Correlation of STX7 with immune infiltration. A A heatmap shows the relationships between STX7 expression and ESTIMATEScore, ImmuneScore, and StromalScore, with circles marking statistically significant correlations (p < 0.05). B A heatmap displays the associations between immune checkpoint markers and STX7 expression across multiple cancer types. Statistical significance is denoted by *, **, and *** for p-values < 0.05, < 0.01, and < 0.001, respectively. C The ssGSEA analysis reveals immune cell infiltration patterns across pan-cancer

We also explored the association between STX7 and various chemokines, receptors, and immune stimulators using TISIDB. The heatmap in Figure S2 shows that STX7 is positively associated with various chemokines, immune receptors, stimulatory factors, and elevated promoter methylation across several cancers. This indicates that STX7 expression modulates the immune microenvironment via chemokine signaling, likely contributing to tumor progression by altering the immune landscape. Additionally, we assessed STX7 expression across various immune cell types. Using the ssGSEA algorithm, we found a significant correlation between STX7 and 24 immune cell types, underscoring its potential role in shaping the immune profile within cancerous environments (Fig. 3C).

Single-cell RNA sequencing analysis in HCC

We included 10 HCC patients with varying tumor-node-metastasis (TNM) stages and hepatitis virus infection statuses. Eighteen distinct cell clusters were identified and annotated based on classical cell-type marker genes, resulting in categories including hepatocytes, endothelial cells, T/NK cells, epithelial cells, macrophages, B cells, fibroblasts, monocytes, and plasma cells (Figs. 4A, B).

Fig. 4

Single-cell and spatial transcriptomic analysis of STX7 expression in HCC. A Heatmaps of the top five genes and functional enrichment analysis for different cell types in single-cell transcriptome. B The UMAP plot illustrates the distribution of distinct cell subtypes within the multicellular environment of HCC patients, with each subtype represented by a unique color. C The expression levels of STX7 in HCC cells. D Pseudotime and trajectory analysis of macrophages and monocytes in HCC. (E) The trend of STX7 expression across pseudotime variation. F Spatial transcriptomic sections showing overlapping patterns of H&E staining, CD14, CD68, and STX7 expression

Elevated STX7 expression in macrophage cells in HCC

In HCC, STX7 exhibits a predominant expression pattern in monocytes and macrophages, particularly in macrophages (Fig. 4C). Pseudotime trajectory analysis further demonstrates the variability in STX7 expression during the progression of the monocyte-macrophage lineage (Figs. 4D, E).

Spatial transcriptomic data confirmed that STX7 expression patterns overlapped substantially with those of the macrophage marker CD14 and CD68 in HCC (Fig. 4F), implied potential co-localization of these genes.

Intercellular communication in macrophages with varying STX7 expression levels

To investigate the regulatory role of STX7 in intercellular communication, we compared STX7 + and STX7- macrophages and used the CellChat tool to analyze differences in signaling interactions. The analysis revealed that STX7 + macrophages, as receivers, showed significantly stronger communication with epithelial cells and fibroblasts than STX7- macrophages. Additionally, as signal senders, STX7 + macrophages showed enhanced interactions with monocytes and T/NK cells, compared to STX7- macrophages (Fig. 5). In several signaling pathways, STX7 + macrophages demonstrated stronger overall signal transmission than STX7- macrophages. These pathways included input signals (ANNEXIN, COMPLEMENT, GAS, PROS, CHEMERIN, IL16) and output signals (GALECTIN, CXCL, BAFF, TNF, IL10) (Fig. 6A). STX7 + macrophages exhibited prominent activity in ligand-receptor (L-R) interactions with endothelial cells, monocytes, and fibroblasts. Specifically, interactions with monocytes and fibroblasts prominently activated the MIF (CD74 + CXCR4) signaling pathway, while communication with endothelial cells resulted in the activation of the C3 (ITGAX + ITGB2) and RARRES2 (CMKLR1) signaling pathways (Fig. 6B). These findings underscore the critical role of STX7 + macrophages in orchestrating intercellular signaling within the immune microenvironment.

Fig. 5

Circle plots showing cell-cell communications of main cell clusters. Each cell cluster acts as a signaling sender or signaling receiver conducting intercellular crosstalk with STX7 + macrophages and STX7- macrophages, respectively

Fig. 6

Regulatory role of STX7 in cell-cell communication. A Overview of incoming and outgoing information flows mediated by various signaling pathways in the main cell clusters. B Communication probabilities of key ligand-receptor pairs that mediate cell-cell interactions between main cell clusters and STX7 + or STX7- macrophages. The color of each dot indicates the probability of communication, while the size of the dot corresponds to the p-value. A lack of corresponding ligand-receptor pairs is indicated by empty meaning, suggesting no communication in that cell

STX7 enhances the progression of hepatocellular carcinoma and modulates macrophage recruitment

Through comprehensive experimental analyses, we confirmed the upregulation of STX7 protein expression in HCC tissues (Fig. 7A-C). Compared to the relatively low expression observed in L-02 cells, STX7 was markedly upregulated in the HCC cell lines (Fig. 7D). The full-length, uncropped Western blot images supporting this result are provided in the supplementary file titled “Uncropped_FullLength_Original_WesternBlots”. Leveraging the naturally high STX7 expression in these HCC cell lines, stable knockdown of STX7 was established in JHH-7 and SNU-475 cells (Fig. 7E). STX7 silencing significantly inhibited HCC cell proliferation, as shown by the CCK-8, EdU, and colony formation assay (Figs. 7F-J). Scratch wound assays further revealed that STX7 knockdown impaired cell migration and invasion (Figs. 7K, L).

Fig. 7

Evaluation of the role of STX7 in regulating the malignancy of hepatocellular carcinoma cells through the NF-κB pathway. STX7 expression in paired HCC tumor and adjacent non-cancerous tissues, as assessed by immunohistochemical staining (A), western blot (B), and qPCR (C) analysis, respectively. D Expression levels of STX7 protein in liver cell lines. E mRNA and protein levels of STX7 in transfected cells. F-J The effect of STX7 knockdown on tumor cell proliferation, assessed using CCK-8, EdU uptake assays, and colony formation assays. (K, L) Tumor cell migration following STX7 knockdown, assessed through a wound healing assay. M Western blot results showed the effects of STX7 knockdown on EMT-and NF-κB-related proteins in JHH-7 and SNU-475 cells

In JHH-7 and SNU-475 cells, STX7 protein downregulation resulted in a marked upregulation of E-cadherin levels and a simultaneous decrease in the expression of N-cadherin, MMP2, and MMP9. Additionally, p-NF-κB p65 expression was significantly reduced following STX7 knockdown, whereas the expression of NF-κB p65 remained largely unchanged. These findings suggest that STX7 may promote the epithelial-mesenchymal transition (EMT) process in HCC cells via the activation of the NF-κB signaling pathway (Figs. 7M). These full-length, uncropped Western blot images are provided in the supplementary file titled “Uncropped_FullLength_Original_WesternBlots”.

STX7 knockdown also led to a substantial reduction in macrophage infiltration (Figs. 8A, B). In vivo experiments demonstrated that STX7 silencing significantly reduced subcutaneous xenograft tumor growth and macrophage infiltration compared to controls (Figs. 8C-G). These findings highlight the critical role of STX7 in promoting HCC cell proliferation, migration, and invasion.

Fig. 8

Investigating STX7-mediated macrophage infiltration and its impact on tumor progression in vivo. A, B Migration of macrophages toward tumor cells following STX7 knockdown in JHH-7 and SNU-475 cells. C Representative images of tumors. D Tumor weights. E Tumor volumes. F, G Immunohistochemical staining of tumor tissues for CD68 and Ki-67. Statistical significance is represented as *P < 0.05, **P < 0.01, ***P < 0.001