PCMT1 expression is increased in liver cancer patients

We analyzed the mRNA expression of PCMT1 in 33 types of cancer, observing that its expression levels varied significantly among different cancers. However, PCMT1 was predominantly upregulated in cancer (Fig. 1A) and PCMT1 expression was associated with higher clinical stage (Additional file 1: Fig. S1). Overall, we discovered that PCMT1 expression was considerably increased in LIHC samples compared to adjacent normal tissues (Fig. 1B). We further analyzed 50 pairs of tumor samples and corresponding adjacent normal tissues from the same patient, finding that PCMT1 expression was significantly elevated in tumor tissues (Fig. 1C). To validate the role of PCMT1 in LIHC treatment, we selected a dataset of LIHC patients who underwent transarterial chemoembolization (TACE) treatment from the Gene Expression Omnibus (GEO) (GSE104580). We first divided HCC patients into two groups according to the level of PCMT1 expression, and our analysis showed that patients with low PCMT1 expression accounted for more of TACE-responsive patients. (Fig. 1D). These results suggest that PCMT1 may play an important role in the progression of liver cancer.

Fig. 1

PCMT1 is highly expressed in LIHC and many other tumors. A Analysis of PCMT1 expression in liver cancer and adjacent normal tissues in the TCGA database. B TCGA database and statistical analyses of PCMT1 expression in 58 pairs of LIHC tissues and adjacent normal tissues. C The mRNA level of PCMT1 in TCGA. The blue and red bar graphs indicate normal and tumor tissues, respectively. D Relationship between TACE therapy reactivity and PCMT1 expression. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

PCMT1 expression was associated with the poor prognosis and clinicopathological factors of LIHC

Our results showed that PCMT1 was significantly upregulated in T3 and T4 stages compared to T1 stage and in stage III compared with stage I (Additional file 1: Fig. S1). It is noteworthy that higher expression of PCMT1 in liver cancer patients is associated with poorer OS and PFS (Fig. 2A, B). We further plotted ROC curves to evaluate the prognostic value of PCMT1, which showed that the AUC values corresponding to 1-year, 3-year, and 5-year survival were 0.641, 0.608, and 0.627, respectively (Fig. 2C). Furthermore, univariate Cox regression analysis identified stage and PCMT1 as prognostic factors for LIHC patients (Fig. 2D), and multivariable Cox regression analysis revealed that both high expression of PCMT1 and advanced tumor stage were independent unfavorable prognostic factors for LIHC patients’ prognosis (Fig. 2E). Our research findings indicate a close correlation between PCMT1 expression and clinical features as well as prognosis of LIHC patients, highlighting its potential as a therapeutic target and prognostic assessment in liver cancer.

Fig. 2

PCMT1 is associated with survival in patients with liver cancer. A The relationship between PCMT1 and OS in liver cancer patients; B the relationship between PCMT1 and PFS in liver cancer patients; C ROC curve of prognostic prediction of liver cancer patients, univariate (D) and multivariate survival analysis (E) combined with clinical information of PCMT1

Differential gene pathway analysis associated with PCMT1

Firstly, we identified differentially expressed genes (DEGs) based on the expression levels of PCMT1, with a total of 2582 genes exhibiting differential expression. The heatmap in Fig. 3A illustrates the relationship between the top 50 DEGs and the expression levels of PCMT1. Furthermore, Gene Ontology (GO) term pathway enrichment analysis, as shown in Fig. 3B, revealed that PCMT1 affects immune functions such as humoral immune response mediated by circulating immunoglobulin, B cell-mediated immunity, and T cell receptor complex. KEGG pathway analysis results demonstrated that DEGs were primarily enriched in neuroactive ligand–receptor interaction, cytokine–cytokine receptor interaction, proteoglycans in cancer, cell cycle, and cell adhesion molecules (Fig. 3C). Subsequently, we performed Gene Set Enrichment Analysis (GSEA) using the KEGG pathway genome. As shown in Fig. 3D, the expression levels of PCMT1 were associated with the neuroactive ligand–receptor pathway, olfactory transduction, and primary bile acid biosynthesis.

Fig. 3

Differential gene analysis of tumor samples with high and low PCMT1 expression. A Heat map of the top 50 differentially expressed genes of PCMT1. B GO analysis of differentially expressed genes. C KEGG analysis of differentially expressed genes. D GSEA analysis of differentially expressed genes

Immune infiltration analysis of PCMT1 in LIHC cancer

The immune microenvironment plays a critical role in tumor progression and treatment. We assessed the impact of differential PCMT1 expression on immune cell infiltration. The results showed that PCMT1 significantly affects immune cell infiltration in tumors (Fig. 4A). Using the ssGSEA algorithm, we further evaluated the correlation between PCMT1 expression and immune cell infiltration, observing a negative correlation between PCMT1 and eosinophil and activated NK cell infiltration, but a significant positive correlation with monocyte and CD4+T cell infiltration (p < 0.05, Fig. 4B–D). The expression of immune checkpoints affects the composition of the entire immune microenvironment; therefore, we further evaluated the relationship between PCMT1 and 33 immune checkpoints in LIHC. The results showed that the PCMT1 level is positively correlated with the levels of various immune checkpoints in LIHC, especially HAVCR2, LAIR1, CD86, and CD274 (Fig. 4E).

Fig. 4

The expression of PCMT1 is associated with immune infiltration. A The relationship between PCMT1 and immune cell infiltration; B the correlation parameters between PCMT1 and various immune cell infiltration; C the correlation between PCMT1 and CD4memoryT cells; D the correlation between PCMT1 and NK cells; E the correlation heat map between PCMT1 and immune checkpoint

PCMT1 was associated with cancer-related gene mutation and drug sensitivity

To explore the expression of PCMT1 and its responsiveness to immune therapy, we employed immune phenotyping (IPS) to evaluate the response to immune checkpoint inhibitors (ICI) treatment. According to the expression of CTLA4 and PD1, the patients were divided into four groups to evaluate the influence of the expression level of PCMT1 on the sensitivity to ICI treatment. Our results showed that high expression of PCMT1 in CTLA4 and PD1 double negative group and in CTLA4 positive and PD1 negative group would reduce the sensitivity to ICI treatment (Fig. 5A). To determine the differences in cancer-associated gene mutations between the PCMT1 high and low-expression groups, we assessed the mutation status of representative genes in each group. PCMT1 expression was not related to tumor mutation burden (Additional file 2: Fig. S2). The general information on representative gene mutations in each group is presented (Fig. 5B, C). From the results, TP53, CTNNB1, TTN, MUC16, and CSMD3 were the top five genes with the highest mutation frequency in the high-expression group, while TTN, CTNNB1, MUC16, TP53, and PCLO were the top five genes with the highest mutation frequency in the low-expression group. Therefore, PCMT1 may affect the mutation status of cancer-related genes.

Fig. 5

PCMT1 affects TP53 mutations and immunotherapy outcomes. A The correlation between PCMT1 expression and ICI treatment was predicted by IPS score, a lower score predicted a lower immunotherapy response rate. B The top30 mutant genes with high expression of PCMT1 were grouped. C The top30 mutant genes with low expression of PCMT1 were grouped. D Analysis of drug sensitive screening for PCMT1

To further investigate drugs that may be effective against high expression of PCMT1, we compared the estimated IC50 levels of 138 chemotherapy drugs or inhibitors. Representative drugs are shown in Fig. 5D. Lower values may indicate higher sensitivity to the drug. We found that BAY 61-3606, BI-2536, 5-fluorouracil, AT-7519, bleomycin, and BMS345541 are potential candidate drugs for treating patients with PCMT1 high expression.

Knockdown of PCMT1 inhibited the growth, migration and increased apoptosis of liver cancer cells

Initially, we conducted Western blotting to examine the protein expression levels of PCMT1 in the tumor tissue and adjacent non-tumor tissue of liver cancer patients. The results revealed a significant increase in PCMT1 expression in the tumor region (Fig. 6A).

Fig. 6

PCMT1 affects the growth and invasion of hepatocellular carcinoma cells. A The expression of PCMT1 in tumor tissue and adjacent to tumors in patients with liver cancer. B The expression of PCMT1 knocked down in two tumor cells. C The effect of PCMT1 knocked down on the growth of tumor cells. D The migration experiment of two tumor cells after PCMT1 knocked down. E, F The flow cytometry analysis of the apoptosis of two liver cancer cells after PCMT1 knocked down and corresponding statistical charts

In vitro experiments demonstrated that the knockdown of PCMT1 significantly inhibited the proliferation of HEPG2 and Hep1-6 cells (Fig. 6B, C). Additionally, the Transwell assay confirmed that the migration ability of the cells was significantly reduced following PCMT1 knockdown (Fig. 6D). Furthermore, flow cytometry analysis revealed a significant increase in cell apoptosis after PCMT1 knockdown (Fig. 6E, F). These findings indicate that PCMT1 may play a vital regulatory role in liver cancer.

RNA sequencing showed that PCMT1 was associated with PI3K pathway and apoptosis

We employed RNA transcriptome sequencing to explore the potential mechanism of PCMT1's role in liver cancer. The results showed that the knockdown of PCMT1 led to the upregulation of 112 genes and the downregulation of 22 genes, Go analysis showed that differentially expressed genes were mainly related to intrinsic component of membrane (Fig. 7A). KEGG analysis of the differentially expressed genes indicated that PCMT1 may affect apoptosis and the PI3K–AKT pathway (Fig. 7B). We validated the expression of apoptosis-related proteins, BAX and BCL-2, following PCMT1 knockdown in two liver cancer cell lines. The results demonstrated a significant increase in the expression of BAX after PCMT1 knockdown, the expression of BCL-2 protein was significantly decreased (Fig. 7C). Additionally, we confirmed that the phosphorylation levels of PI3K and AKT were reduced following PCMT1 knockdown. Lastly, we examined EMT-related proteins and found that PCMT1 knockdown could decrease EMT in liver cancer cells (Fig. 7D).

Fig. 7

PCMT1 affects the PI3K–AKT pathway. A Volcano map of differentially expressed genes obtained by RNA sequencing after PCMT1 knockdown. B KEGG analysis of differentially expressed genes. C The effect of PCMT1 knockdown on the levels of apoptosis-related proteins and quantitative analysis of protein levels. D The effect of PCMT1 knockdown on the phosphorylation of PI3K, AKT, the expression of EMT-related proteins and quantitative analysis of protein levels

Knockdown of PCMT1 inhibited tumor growth and promoted tumor apoptosis

We further studied the effect of PCMT1 on tumor growth in vivo, and the results showed that the tumor growth curve was significantly slower and the tumor weight was significantly lower in the mice with PCMT1 knockdown (Fig. 8A–C). After further staining of tumor tissues, it was found that knockdown PCMT1 could significantly increase the apoptosis of tumor region. This was further confirmed by caspase-3 staining, and HE staining was also performed, which showed that apoptosis of the tumor area was very obvious after knockdown of PCMT1 (Fig. 8D).

Fig. 8

PCMT1 affects tumor growth and apoptosis. A Tumor taken from mice in groups with or without PCMT1 knockdown. B The tumor volume from mice in two groups over time; C comparison of tumor weight between two groups with or without PCMT1 knockdown; D fluorescence staining of TUNEL and caspase-3 and corresponding HE staining in tumor samples of mice in two groups

PCMT1 affects the infiltration and function of immune cells in tumor area

Bioinformatics immunoassay results showed that PCMT1 could affect immune infiltration in the tumor region, so we further examined the immune cell infiltration in the tumor region of mice in the two groups with or without PCMT1 knockdown. We detected the infiltration of CD4 and CD8T cells in the tumor area by flow cytometry, and the results showed that the infiltration of CD8+T cells was significantly increased after PCMT1 knockdown (Fig. 9A–C). The immunofluorescence staining of tumor sections showed that the expression of CD86 was significantly increased after PCMT1 knockdown, while the expression of CD206 was decreased (Fig. 9D). This may be related to the promotion of M1 polarization of macrophages, thus promoting anti-tumor immunity, which may also be the possible reason for the slower tumor growth after PCMT1 knockdown.

Fig. 9

PCMT1 affects immune cell infiltration and macrophage phenotype in the tumor area. A The effect of PCMT1 knockdown on the infiltration of CD4+T cells and CD8+T cells in tumor area; B statistical analysis of CD8+T cell infiltration in tumor area under different treatments; C statistical analysis of CD8+T cell infiltration in tumor area under different treatments; D fluorescence staining of CD11b and macrophage subtype markers CD86 and CD206 in two groups of tumors