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Expression, DNA methylation pattern and transcription factor EPB41L3 in gastric cancer: a study of 262 cases

Cell Communication and Signaling volume 22, Article number: 470 (2024) Cite this article

Abstract

Purpose

DNA methylation prominently inactivates tumor suppressor genes and facilitates oncogenesis. Previously, we delineated a chromosome 18 deletion encompassing the erythrocyte membrane protein band 4.1-like 3 (EPB41L3) gene, a progenitor for the tumor suppressor that is differentially expressed in adenocarcinoma of the lung-1 (DAL-1) in gastric cancer (GC).

Methods

Our current investigation aimed to elucidate EPB41L3 expression and methylation in GC, identify regulatory transcription factors, and identify affected downstream pathways. Immunohistochemistry demonstrated that DAL-1 expression is markedly reduced in GC tissues, with its downregulation serving as an independent prognostic marker.

Results

High-throughput bisulfite sequencing of 70 GC patient tissue pairs revealed that higher methylation of non-CpGs in the EPB41L3 promoter was correlated with more malignant tumor progression and higher-grade tissue classification. Such hypermethylation was shown to diminish DAL-1 expression, thus contributing to the malignancy of GC phenotypes. The DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-CdR) was found to partially restore DAL-1 expression. Moreover, direct binding of the transcription factor CDC5L to the upstream region of the EPB41L3 promoter was identified via chromosome immunoprecipitation (ChIP)-qPCR and luciferase reporter assays. Immunohistochemistry confirmed the positive correlation between CDC5L and DAL-1 protein levels. Subsequent RNA-seq analysis revealed that DAL-1 significantly influences the extracellular matrix and space-related pathways. GC cell RNA-seq post-5-Aza-CdR treatment and single-cell RNA-seq data of GC tissues confirmed the upregulation of AREG and COL17A1, pivotal tumor suppressors, in response to EPB41L3 demethylation or overexpression in GC epithelial cells.

Conclusion

In conclusion, this study elucidates the association between non-CpG methylation of EPB41L3 and GC progression and identifies the key transcription factors and downstream molecules involved. These findings enhance our understanding of the role of EPB41L3 in gastric cancer and provide a solid theoretical foundation for future research and potential clinical applications.

Plain English summary

The EPB41L3 gene, frequently exhibiting haplotype deletions and reduced expression in gastric cancer tissues, points to its potential role as a tumor suppressor. However, tumor suppressor genes are not only influenced by genomic deletions but also by their methylation status. Our study highlights the significantly lower expression of EPB41L3 in gastric cancer compared to adjacent non-cancerous tissues across 262 patients. We also discovered that elevated non-CpG island methylation of EPB41L3 correlates strongly with tumor malignancy progression, based on the analysis of 70 paired gastric cancer samples. Moreover, we identified CDC5L as a crucial transcription factor interacting with the EPB41L3 promoter. Integrative analyses of transcriptomic and single-cell sequencing data further revealed that AREG and COL17A1 are key downstream molecules regulated by DAL-1, with their expression tightly controlled by EPB41L3 methylation and expression levels. These insights enhance our understanding of EPB41L3’s role in gastric cancer and could open new avenues for targeted therapies.

Introduction

Gastric cancer (GC), one of the major malignant tumors, is the fourth leading cause of cancer death worldwide [1]. The great economic and health burden caused by GC urges researchers to explore the molecular mechanisms underlying its pathogenesis and to identify potential therapeutic targets against this disease.

Chromatin was the earliest identified target for cancer treatment. The alteration of DNA methylation is a typical chromosome change related to oncogenesis [2]. The genome of tumors is usually accompanied by whole hypermethylation but hypomethylation of CpG islands, especially the CpG islands of tumor suppressor genes. Therefore, an inhibitor of DNA methylation, 5-Aza-CdR, which reactivates the expression of suppressor genes, has been clinically employed for antitumor purposes [3, 4].

Multiple variations drive the evolution of GCs, which undergo multiple pathways and multistage processes. Accumulating evidence has shown that inactivation of tumor suppressor genes, which frequently manifests as alterations in DNA modifications, such as increased methylation levels [3, 4], is one of the main molecular mechanisms involved in the occurrence and development of GC [5, 6].

In our previous study, we identified a deletion region of the chromosome 18 short arm (18p11.3) [7, 8], in which the EPB41L3 gene is located. DAL-1, an EPB41L3-encoding protein, belongs to the protein 4.1 superfamily [9] and has been shown to be frequently absent in a variety of cancer types, including lung cancer, breast cancer, ovarian cancer and prostate cancer [10,11,12,13,14]. The major inactive mechanisms of EPB41L3 usually include loss of heterozygosity (LOH) on chromosome 18p or hypermethylation in its CpG island [15]. Studies have shown that EPB41L3 acts as a tumor suppressor that attenuates cell migration and the epithelial mesenchymal transition (EMT) process and promotes apoptosis [10, 16]. Our previous data indicated that DAL-1 plays a crucial role in the genesis and development of GC [17]. However, the correlation between the expression and methylation status of EPB41L3 and the clinical prognosis of gastric cancer patients and the underlying mechanism of DAL-1 in support of its suppressive role in GC remain to be clarified.

Therefore, the aim of this study was to investigate the relationships of EPB41L3 gene expression and promoter methylation with the clinical progression of GC. Additionally, this study sought to identify the transcription factors and downstream regulatory molecules associated with EPB41L3. This research may offer deeper insight into the role of the core tumor suppressor gene EPB41L3 in gastric cancer.

Materials and methods

Ethics, consent, and permissions

All patients provided signed informed consent and passed the review of the medical ethics committee of Harbin Medical University (KY2018014).

Tissues and cell culture

AGS and HGC-27 gastric cancer cell lines are mycoplasma free and have been authenticated via STR (or SNP) profiling within the last three years.

Targeted high-throughput bisulfite sequencing.

Data processing [18].

Gene enrichment analysis [19,20,21].

Public single-cell mRNA sequencing data processing [22].

Immunohistochemistry.

Western blot.

Dual-luciferase reporter assay.

Chromosome immunoprecipitation (ChIP).

MTS proliferation.

Colony formation assay.

Wound healing assay.

Transwell assay.

Quantitative PCR (qPCR).

Cell apoptosis assay and cell cycle analysis.

RNA sequencing.

Statistical analysis

Owing to the article word limit, method details are included in the Supplementary Methods.

Result

The decreased expression of DAL-1 in patients with GC is associated with poor prognosis

The RNA-Seq data of 415 GC and 35 adjacent non-cancerous tissues from the TCGA database revealed that the DAL-1 expression level in GC tissues was significantly lower than that in normal gastric tissues (Fig. 1A). As expected, DAL-1 levels were significantly lower in GC tissues than in adjacent tissues (Fig. 1B, n = 132, cancerous and adjacent noncancerous paired tissues). Furthermore, correlation analysis of DAL-1 expression and clinicopathological features from 262 GC patients (132 paired tissues and 130 GC patient tissues with survival data) demonstrated that DAL-1 tended to be higher in T1 than in T2 and T3 patients (***P < 0.001) (Fig. 1C) and to be lower in samples with lymph node metastasis (P = 0.008) (Fig. 1D, Supplementary Table 1). Data from GSE13911 [23] also revealed higher DAL-1 expression in normal gastric tissues than in GC tissues (Fig. 1E). Moreover, we found that patients with lower DAL-1 levels had shorter overall survival (OS) than did those with higher DAL-1 levels (Fig. 1F) in a cohort of 130 GC patients. The Cox proportional hazards model multivariate analysis also revealed that a low DAL-1 level was significantly correlated with shorter OS (HR, 0.385; 95% CI, 0.204–0.628; ***P < 0.001), which suggested that DAL-1 could be an independent prognostic factor for poor prognosis (Fig. 1G).

Fig. 1
figure 1

Decreased DAL-1 expression is correlated with poor prognosis in patients with GC. A. Expression profile of DAL-1 in GC tissues on the basis of TCGA data; Student’s t test. B. DAL-1 expression in GC tissues by IHC (n=132), Wilcoxon test. Magnification ×20, scale bar=50 μm. C. Expression of DAL-1 in T1~T4 GC tissues, Wilcoxon test. D. Proportion of patients with high DAL-1 expression and low DAL-1 expression in the N0 and N≥1 groups. E. Expression of DAL-1 in GC and normal tissues in GSE13911, Wilcoxon test. F. Kaplan‒Meier survival analyses according to DAL-1 expression in GC patients (n=130). G. Univariate and multivariate Cox regression analyses of different prognostic factors in patients with GC. **, P<0.01; ***, P<0.001

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Increased non-CpG methylation in the EPB41L3 promoter correlates with advanced cancer progression

Hypermethylation of the promoter region of tumor suppressor genes contributes to low gene expression levels. Analysis of data from The Cancer Genome Atlas (TCGA) database revealed a significant negative correlation between DAL-1 mRNA expression and the methylation status of 10 CG sites in its promoter region in GC cells (Fig. 2A).

Fig. 2
figure 2

The methylation level of EPB41L3 is significantly greater in GC tissues than in adjacent noncancerous tissues. A. Association between DAL-1 mRNA expression level and promoter methylation at 10 CG sites. B. CpG island prediction via MethPrimer 2.0. C. Differential expression analysis of DAL-1 in 132 pairs of GC and noncancerous tissues. Samples for bisulfite sequencing (green rectangles), high DAL-1 expression in GC tissue (red rectangles), and low DAL-1 expression in GC tissue not selected for sequencing (black rectangles) were selected. D. Methylation percentage of the EPB41L3 promoter region in islands 1 and 2. CpG, CHG and CHH are the different sequence motifs of methylation (whereby H can be either A, T or C). Each point represents the methylation percentage of all cytosine bases in the relative context of each tissue. E. Methylation percentage of the EPB41L3 promoter region at each CpG site in islands 1 and 2 of GC and normal gastric tissue. *P<0.05, **P<0.01, ***P<0.001

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Methylation analysis of the EPB41L3 promoter region focused on a 1 kb region surrounding the transcription start site, which was identified to contain two CpG islands (designated island 1 and island 2) through CpG island prediction via MethPrimer 2.0 online (Fig. 2B). Owing to the sparse CpG distribution of island 2, methylation primers were designed in the regions 1 kb upstream and 1.5 kb downstream of the island. High-throughput bisulfite sequencing was subsequently performed on 70 selected tissues with significantly reduced DAL-1 expression out of the 132 GC tissues (Fig. 2C). Figure 2D shows a significantly greater percentage of methylated cytosines at both island 1 and island 2 in GC tissues than in adjacent non-cancerous tissues. Specifically, in the CHG, CHH context of island 1 and CG context of island 2, cytosine methylation levels were significantly higher in GC tissues than in noncancerous tissues. We analyzed the methylated cytosines in the CG context and highlighted differences in the methylation patterns of the EPB41L3 promoter region between GC and normal gastric tissues. Methylation levels at many sites within island 1 were relatively high in GC tissues (Fig. 2E).

We also found an increase in the average methylation levels of island 1 and island 2 in the three GC cell lines compared with those in the normal gastric mucosal epithelial line GES-1 (Supplementary Table 2). Furthermore, AGS cells presented the highest differentially methylated levels of the EPB41L3 gene promoter region (Supplementary Table 3).

We subsequently analyzed the relationship between the methylation level of the CpG island cytosine in the promoter region of EPB41L3 in GC tissues and clinical factors. The samples were divided into a hypermethylation group and a hypomethylation group on the basis of the standard in Joseph R E’s work [24]. As shown in Table 1, in the CHH motif of island 1, hypermethylated cytosine is positively correlated with tumor size, poor differentiation and the number of lymph node metastases. Table 1 shows similar results: the hypermethylation of cytosine in the CHG and CHH motifs of island 2 was positively correlated with tumor size, poor differentiation, and the number of lymph node metastases. The proportion of cytosine hypermethylation in CG and CHG motifs increases with increasing clinical stage. In addition, the proportion of CHG and CHH hypermethylation in CpG island 2 in patients with intestinal GC is significantly greater than that in patients with diffuse GC. Collectively, these findings demonstrate that hypermethylation of the EPB41L3 gene promoter is closely related to cancer progression and the histopathological subtypes of GC.

Table 1 Correlation between DAL-1 islands methylation level and clinicopathological features in GC
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The transcription factor cell division cycle 5-like protein (CDC5L) promoted EPB41L3 gene transcription

Some proteins use a negative/positive feedback mechanism to ensure their own expression at a steady level. To identify the transcription factor of DAL-1 that potentially functions as a feedback loop, we predicted the interaction proteins of DAL-1 from the NCBI, UniProt, STRING, BiOGRID, and PubMed databases and enriched them via GO and KEGG analyses. As shown in Fig. 3A-B, the enriched proteins were involved mainly in the regulation of cell biological processes such as the cell cycle, the tight junction functional pathway and the Wnt signaling pathway. Among these enriched proteins, only three proteins, CDC5L, NFIL3 and KLF13, had transcription factor activity. Moreover, bioinformatic prediction via the Jaspar database revealed that CDC5L, NFIL3 and KLF13 had 7, 1 and 0 binding sites, respectively, to EPB41L3 (Supplementary Fig. 1A). These findings suggest that CDC5L might have transcriptional activation potential on EPB41L3.

Fig. 3
figure 3

Identification of the transcription factor CDC5L as a regulator of EPB41L3. A. Representative pathways acquired through GO analysis of the interacting genes upstream and downstream of EPB41L3. *, P<0.05. B. Genes enriched in KEGG pathways among all the interacting proteins and the upstream and downstream proteins of DAL-1. C. Seven pairs of primers spanning the region 3 kb upstream and 1 kb downstream of the gene TSS of the EPB41L3 gene. D. Quality control of the ChIP assay by western blot showing IP-specific protein‒DNA binding. E. CDC5L binding to the EPB41L3 promoter, as determined by ChIP‒PCR. ND, not detected. F. CDC5L activated EPB41L3 transcription according to a luciferase reporter assay. G. Immunohistochemical validation of the positive correlation between CDC5L and DAL-1 expression. *, P<0.05. H. CDC5L was upregulated in GC tissues, as shown by immunohistochemistry analysis. ***, P<0.001. n=129. I. Kaplan‒Meier survival analysis of GC patients. n=119, P=0.43

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We subsequently designed 7 pairs of primers spanning the region 3 kb upstream and 1 kb downstream of the gene transcription start site (TSS) of the EPB41L3 gene, and primer 2 was excluded from the PCR assay (Fig. 3C, Supplementary Fig. 1B & Supplementary Table 4). The ChIP assay results revealed that CDC5L binding sites are enriched in regions amplified by primer 5 and primer 6 (Fig. 3D-E & supplementary Fig. 1C). These findings suggest that the CDC5L binding site of EPB41L3 is in the region 1589–1232 bp upstream of the transcription start site. In the luciferase assay, the promoter region 1169–1719 bp upstream of EPB41L3 was divided into 3 parts and inserted into the pGL3 vector, which were named pEPB41L3-1, pEPB41L3-2 and pEPB41L3-3, respectively. All these vectors were subsequently transfected with a pTT5 eukaryotic expression vector containing the CDS region of CDC5L. The results confirmed that CDC5L could activate the transcription of the EPB41L3 gene through binding to the promoter region 1169–1719 bp upstream of EPB41L3 (Fig. 3F, Supplementary Table 5). Immunohistochemical assays revealed that the expression level of CDC5L in the GC tissues of our cohort was significantly greater than that in the adjacent non-cancerous tissues (Fig. 3G). In addition, the protein expression levels of CDC5L and DAL-1 were positively correlated (Fig. 3H, r = 0.15, *P < 0.05), suggesting that CDC5L might positively regulate the expression of DAL-1. However, there was no significant correlation between CDC5L protein expression and clinicopathological parameters (Supplementary Table 6). No significant difference in overall survival (OS) was found between patients with gastric cancer (GC) and patients with CDC5L expression, as shown in Fig. 3I.

Downstream pathways regulated by DAL-1 or EPB41L3 hypermethylation

We established AGS-overexpressing DAL-1 and HGC-27 DAL-1 knockdown models and analyzed the differentially expressed genes (DEGs) following RNA sequencing (Supplementary Fig. 2A-D). To identify the genes positively regulated by DAL-1, we took the intersection between the genes upregulated after DAL-1 overexpression and those downregulated by DAL-1 silencing and obtained EPB41L3 and TMEM156 (Fig. 4A). Twenty-eight gene ontology terms were positively regulated by DAL-1, which were at the intersection of the genes whose expression was upregulated after DAL-1 overexpression and those whose expression was downregulated by DAL-1 silencing (*P < 0.05) (Supplementary Fig. 2E). Thirteen gene ontology terms were negatively regulated by DAL-1, which were the common differential expression pathways of the genes downregulated after DAL-1 overexpression and those upregulated by DAL-1 silencing (*P < 0.05) (Supplementary Fig. 2F). Interestingly, altering DAL-1 expression, whether by upregulation or downregulation, impacts these three pathways, including the regulation of mitophagy, the extracellular space and the extracellular matrix (Fig. 4B).

Fig. 4
figure 4

Genes and ontologies regulated by DAL-1 or EPB41L3 hypermethylation in GC cell lines. A. Genes positively regulated by DAL-1. *, P<0.05; ***, P<0.001. B. A Venn diagram describing the four GO term sets of upregulated genes and downregulated genes in the ov-DAL-1 group and sh-DAL-1 group. The table on the right side describes the 3 GO ontologies in the intersection and the DE gene names of these terms in the four sets. C. FPKM values of DAL-1 in the control and 5-Aza-CdR-treated groups. ***, P<0.001. D. Western blot analysis verified that the expression of DAL-1 was downregulated after 5-Aza-CdR treatment. E. Differentially expressed genes after 5-Aza-CdR treatment. F. GO analysis of the hub ontologies and hub DE genes positively regulated by DAL-1 demethylation. G. Venn diagram showing the GO terms negatively regulated by EPB41L3 demethylation. The table on the right side shows the hub ontologies and genes negatively regulated by DAL-1 demethylation

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According to our findings, EPB41L3 is often hypermethylated, leading to reduced DAL-1 expression in gastric cancer. To examine the effects of EPB41L3 methylation on its downstream genes, AGS cells were treated with 5-Aza-CdR to reduce EPB41L3 methylation, and the DAL-1 expression level was increased (Fig. 4C-D). RNA sequencing revealed 409 upregulated genes and 128 downregulated genes (Fig. 4E). Subsequent analysis revealed 25 upregulated and 6 downregulated genes common to DAL-1 overexpression and 5-Aza-CdR treatment (Supplementary Table 7). Gene ontologies affected by DAL-1 demethylation were investigated, with 79 terms shared between EPB41L3 demethylation and DAL-1 overexpression (Supplementary Fig. 2G), highlighting key terms related to extracellular regulation, mitophagy, and cell proliferation. Thirteen Hub genes, such as REG4, AREG, and COL17A1, were identified (Fig. 4F).

By analyzing the terms negatively regulated by EPB41L3 demethylation, 48 terms were obtained after 5-Aza-CdR treatment and DAL-1 downregulation (Supplementary Fig. 2H). After jointly examining the negatively regulated ontologies following DAL-1 downregulation, five pathways were collectively identified, including positive regulation of mitophagy, regulation of phosphatidylinositol 3-kinase activity, lipid kinase activity, phospholipid metabolic process and cellular carbohydrate metabolic process. Two hub genes, HKDC1 and RP11-227H15.5, were identified (Fig. 4G).

DAL-1 downstream genes AREG and COL17A1 verified by single-cell sequencing

Gene expression profiling of gastric cancer tissue revealed a mixed state of gene expression in both gastric cancer cells and their microenvironmental cells. To validate the significant correlation between DAL-1 expression levels and hub genes in gastric cancer epithelial cells, we utilized single-cell sequencing data from 6 GC and 4 normal tissues (GSE183904) [22] to analyze the expression profiles of the 15 hub genes across various cell types. As shown in Fig. 5A&B, DAL-1 was predominantly expressed in gastric cancer epithelial cells, dendritic cells, fibroblasts, and endothelial cells. Notably, the positive cell ratio and expression levels of the AREG and COL17A1 genes were significantly greater in gastric cancer tissues than in normal gastric tissues, which was consistent with the trend of DAL-1 expression (Fig. 5A-C). Conversely, other hub genes presented the expected expression trends with DAL-1 in other types of cells within the tumor microenvironment, such as fibroblasts and dendritic cells (Supplementary Fig. 3A-C). Survival analysis revealed that low expression of AREG was associated with poor prognosis in GC patients. Similarly, low expression of COL17A1 served as a prognostic indicator for adverse outcomes in intestinal-type GC patients (Fig. 5D).

Fig. 5
figure 5

Single-cell sequencing data revealed the hub genes regulated by the upregulation or demethylation of DAL-1. A. Combined U-MAP plot showing the expression levels of DAL-1, AREG and COL17A1 in clusters of all epithelial and nonepithelial cells from 6 GC tissues and 4 adjacent non-cancerous tissues analyzed via Seurat. B. Comparison of the ratios of DAL-1-, AREG- and COL17A1-positive cells in each cluster in GC and adjacent non-cancerous tissues. C. Comparison of DAL-1, AREG and COL17A1 expression levels in each cluster of cells in GC and adjacent non-cancerous tissues. D. Kaplan‒Meier overall survival curves of patients with GC grouped by the gene signature expression of AREG and COL17A1. E. The regulatory pathway of EPB41L3 in GC progression

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Discussion

Recent research findings have indicated a frequent decrease in DAL-1 expression across various tumor types [25,26,27], which is consistently linked with unfavorable prognoses [28]. Our previous investigations underscored the importance of DAL-1 as a pivotal tumor suppressor in GC [7, 8, 29, 30]. Confirming its relevance, we observed diminished DAL-1 expression in 132 pairs of GC tissues, which was correlated with dismal prognoses, depth of gastric infiltration, and lymph node metastasis within our study cohort. Furthermore, in vitro studies revealed that EPB41L3 downregulation exacerbated the aggressive behavior of GC cells, supporting the tumor suppressor role of DAL-1 in GC [15]. Notably, our study identified low DAL-1 expression as an independent prognostic indicator impacting poor outcomes, suggesting its potential utility as a molecular marker for assessing GC metastasis and prognosis. The outcomes underscore the tumor-suppressive function of EPB41L3 in GC.

Cancer cells display genome-wide DNA hypomethylation and site-specific hypermethylation [31]. Hypermethylation in cancer silences tumor suppressor genes, driving oncogenesis [32]. In our study, DNA methylation contributed to EPB41L3 gene inactivation in GC. Higher methylation levels are observed in EPB41L3 promoter regions in GC tissues, with a negative correlation with lower mRNA expression. Notably, higher methylation of non-CpGs in the EPB41L3 promoter is correlated with larger tumor size, poorer tissue differentiation, stronger tumor metastasis capability, and worse clinical staging. Although non-CpG methylation is mostly observed in stem cells and is rare in developed cells, an increasing number of studies have shown that its alterations are common in cancers and can affect gene expression [33]. For example, the methylated motif C(m)C(A/T)GG of the B-cell-specific B29 gene promoter suppresses the assembly of the DNA‒protein complex and inhibits the binding of the transcription factor EBF to decrease gene expression [34]. Notably, among the 70 GC tissues examined, 35 (50% of the total) presented high levels of methylation at islands 1 and 2 of the EPB41L3 gene promoter, leading to the downregulation of EPB41L3 gene expression. A study in oropharyngeal cancer also demonstrated that EPB41L3 methylation of oral gargle was significantly (P < 0.0001) higher among oropharyngeal cancer cases (n = 101) compared to controls (n = 101). The ROC for EPB41L3 methylation alone indicating an area under the curve (AUC) of 0.738 [26]. However, five non-truncating mutations in the EPB41L3 gene have been detected in tumor tissues and corresponding blood samples from patients with meningioma [35, 36], indicating that other mechanisms may be involved in the functional inactivation of the EPB41L3 gene. Therefore, in future research, a comprehensive mechanistic understanding of EPB41L3 silencing in GC development will be necessary.

The transcription factor CDC5L regulates the expression of the EPB41L3 gene by binding to its promoter region. Although CDC5L has been better studied as an RNA-binding protein involved in pre-mRNA splicing processes, it is also a DNA-binding protein and a transcription factor that prefers a core ‘ANCA’ motif flanking TTA/TAA of DNA [37, 38]. As a transcription factor, CDC5L has been reported to bind to the hTERT promoter region in colorectal cancer, thereby promoting tumor growth and proliferation [39]. CDC5L is also the transcription factor of WNT7B, a critical ligand of the Wnt/β-catenin signaling pathway in lung adenocarcinoma [40]. Our study demonstrated that CDC5L could bind to the upstream promoter region of EPB41L3, which spans 1169–1719 bp from the transcription start site. Interestingly, the binding site is the nonmethylated region of the EPB41L3 promoter. This result suggested that the transcription factor bound to the nonmethylation region of its target gene and reversed the methylation of its DNA flanking sequence [41, 42].

With respect to the function of DAL-1, we found that TMEM156 expression was positively correlated with DAL-1 expression. TMEM156 belongs to the transmembrane protein (TMEM) family. Although little is known about its function, a bioinformatics study in head and neck squamous cell carcinoma has proposed that the overexpression of TMEM156 is associated with immune cell mobilization and better survival rates [43]. GO enrichment revealed the specific role of DAL-1 in exerting its suppressive effect on GC. DAL-1 can negatively regulate cell migration and movement. These results are supported by Yajie Zhang’s study, which revealed a significant increase in migration and invasion after DAL-1 knockdown [10]. In addition, our study revealed that DAL-1 negatively regulates phosphatidylinositol 3-kinase activity, lipid metabolic processes, membrane depolarization and biological quality, all of which are classic oncogenic biological processes.

There are still some limitations in the present study. The gastric cancer samples in this study were obtained from a single center, which may introduce selection bias. Additionally, the mechanisms by which DAL-1 regulates AREG and COL17A1 to influence the tumor microenvironment and malignant phenotype require further elucidation.

Interestingly, whether DAL-1 levels undergo positive or negative changes, both the extracellular matrix and the extracellular space are significantly regulated. Studies have shown that the DAL-1 protein participates in the interaction between cells and the extracellular matrix [44]. It contributes to the reconstruction of the microenvironment by interacting with the extracellular matrix. Single-cell sequencing revealed the hub genes AREG, COL17A1, and REG4, which play roles in the cell matrix, act as suppressors and are positively correlated with DAL-1. Research has shown that AREG suppresses migration by reducing the activity of matrix metalloproteinases, which act as drill bits for tumor metastasis [45, 46]. In epithelial cancers, COL17A1 misexpression is associated with increased invasion [47, 48].

Overall, we showed that GC tissues often present high methylation of the EPB41L3 promoter region and low expression of EPB41L3, which is associated with a poor prognosis in GC patients. Increased methylation of non-CpG sites in the EPB41L3 promoter is associated with characteristics of advanced tumor progression, such as larger tumor size, poorer tissue differentiation, enhanced metastatic potential, and more advanced clinical staging. Additionally, the transcription factor CDC5L is positively correlated with DAL-1 binding to the promoter region of EPB41L3. The overexpression or demethylation of EPB41L3 could regulate the suppressor genes AREG and COL17A1 and ultimately suppress the malignant phenotype of GC (Fig. 5E).

Data availability

The analysis and statistical data of high-throughput bisulfite sequencing of 70 selected tissues with significantly reduced DAL-1 expression among the 132 GC tissues are available from the website https://ngdc.cncb.ac.cn/omix/preview/FNBWEmOs.

Abbreviations

GC:

Gastric cancer

5-Aza-CdR:

5-Aza-2’-deoxycytidine

ChIP:

Chromosome immunoprecipitation

EPB41L3:

Erythrocyte membrane protein band 4.1-like 3

DAL-1:

Differentially expressed in adenocarcinoma of the lung-1

EMT:

Epithelial mesenchymal transition

LOH:

Loss of heterozygosity

qPCR:

quantitative PCR; OS: Overall survival

TCGA:

The Cancer Genome Atlas; CDC5L: Cell division cycle 5-like protein

TSS:

Transcription start site

DEG:

Differentially expressed genes

NSCLC:

Non-small cell lung cancer

DFS:

Disease-free survival

TMEM:

Transmembrane proteins

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Acknowledgements

The results here are partially based upon data generated by TCGA Research. We truly appreciate Mr. Peng Liu, Ms. Xilin Cui, Mr. Guohua Ji, and Mr. An Liu for their assistance in the experimental platform services.

Funding

National Natural Science Foundation of China General Program (No. 30572092).

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Author notes

  1. Mengdi Cai and Haonan Guo contributed equally to this work.

Authors and Affiliations

  1. Scientific Research Centre, The Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Nangang District, Harbin, 150081, China

    Mengdi Cai, Haonan Guo, Dong Wang, Tie Zhao & Jingcui Yu

  2. Key Laboratory of Preservation of Human Genetic Resources and Disease Control in China, Harbin Medical University, Ministry of Education, Harbin, China

    Mengdi Cai, Xiao Liang, Jiaqi Li, XiaoBo Cui, Songbin Fu & Jingcui Yu

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  1. Mengdi Cai
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Contributions

Mengdi Cai: Conceptualization, Data Curation, Writing -Original Draft and Writing – Review & Editing; Haonan Guo: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation and Visualization. Dong Wang and XiaoBo Cui: Writing – Review &Editing; Tie Zhao: Data Curation and Software; Jiaqi Li: Validation and Writing – Review &Editing; Xiao Liang: Project Administration and Software. Songbin Fu: funding acquisition, resources and supervision; Jingcui Yu: conceptualization, methodology, resources, supervision and writing – review & editing. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Jingcui Yu.

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Ethics approval and consent to participate

The study protocol and all experiments with human samples were approved by the Investigation Ethical Committee of the Second Affiliated Hospital of Harbin Medical University. All patients provided informed consent.

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Not applicable.

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N/A.

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The authors declare no competing interests.

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Cai, M., Guo, H., Wang, D. et al. Expression, DNA methylation pattern and transcription factor EPB41L3 in gastric cancer: a study of 262 cases. Cell Commun Signal 22, 470 (2024). https://doi.org/10.1186/s12964-024-01849-7

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Keywords

Cell Communication and Signaling

ISSN: 1478-811X