To draw the genome-wide distribution and remodeling characteristics of H3K27me3 silencers in signet-ring cell carcinoma of the stomach (SRCC) through epigenetic sequencing technology, and to investigate their roles in transcriptional regulation in order to elucidate the regulatory mechanism of SRCC malignant progression.

The study was conducted on 35 gastric samples obtained by gastroendoscopic biopsy (15 normal and 20 SRCC tissues) from Department of Gastroenterology of Army Medical Center of PLA between January 2021 and December 2023. Multi-omics analyses, including assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), cleavage under targets and tagmentation (CUT&Tag) and transcriptome sequencing (RNA-seq), were performed to identify chromatin accessibility, H3K27me3 silencer regions, and transcriptional changes, with aid of Illumina NovaSeq 6000. H3K27me3 related differentially expressed genes (|Log₂FC| >1, FDR<0.05) were screened using DESeq2. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were employed to analyze the enrichment function, and Homer was employed to identify transcription factor motifs. A regulatory network was constructed using Cytoscape, and then validated using immunohistochemistry to explore its regulatory mechanism.

H3K27me3 silencers were primarily located in distal intergenic regions (37.06%) in SRCC. Compared with the normal tissues, SRCC showed a significant reduction in H3K27me3 silencer signals (95%CI: 1.34~2.30, P=0.007) with 6257 lost sites (FDR<0.01). Integrating CUT&Tag and RNA-seq revealed 380 up-regulated immune-related genes, particularly in T cell receptor signaling (OR=4.2, 95%CI: 2.8~6.3, P=0.002). Immunohistochemistry confirmed elevated expression of transcription factor EHF (P<0.05).

There is the remodeling of H3K27me3 silencers in SRCC, and EHF may potentially play a crucial role in the SRCC malignant progression.

To identify the enhancer profile marked by histone H3K27ac modification in high-grade intraepithelial neoplasia (HGIN) in order to reveal the novel regulatory mechanism of HGIN pathogensis.

Gastric tissue samples were collected from Department of Gastroenterology of Army Medical Center of PLA between June 2022 and June 2023, including 14 normal gastric tissues (Nor group), 31 HGIN tissues (HGIN group) and 17 gastric cancer tissues (GC group). Cleavage under targets and tagmentation (CUT&Tag) technique was employed to capture enhancer regions modified by histone H3K27ac. Multi-omics analysis was performed to identify HGIN-specific active enhancers and their potentially regulated genes. Immunohistochemical profiling was performed to assess differential expression of the gene of interest across clinically stratified specimens, combined with CRISPR-dCas9-mediated ablation of active enhancers to monitor the gene of interest transcriptional dynamics and validate enhancer-mediated regulatory mechanisms.

Epigenomic sequencing obtained the data with excellent quality, and indicated that obvious remodeling was observed in H3K27ac enhancers in HGIN and GC groups (P<0.05), though no significant difference in the genome-wide distribution of H3K27ac modification among the 3 groups. Combining transcriptome data revealed that enhancer remodeling may up-regulate the expression of the proliferation-related target gene, CD24, in the HGIN tissue; while, inhibiting enhancer activity can notably reduce CD24 expression level (P<0.05). Immunohistochemical assay displayed a positive correlation between the expression levels of CD24 and Ki-67 (P<0.001).

The remodeling of H3K27ac enhancer represents a significant epigenetic feature of the transformation from normal condition to HGIN. Remodeling of H3K27ac enhancer up-regulates CD24, which may facilitate the abnormal proliferation of gastric epithelial cells.

To analyze clinical characteristics of mesenchymal-subtype gastric cancer (Mes-GC) by integrating multi-omics data and explore the characteristics of tumor cells and microenvironment associated with the risk for recurrence.

Gastric tumor tissue samples were collected from the patients who visited Department of Gastroenterology of Army Medical Center of PLA from January 2022 to December 2023. Transcriptome and genome sequencing were applied for these tissue samples, including 19 cases of diffuse-type gastric cancer, 22 cases of intestinal-type gastric cancer, and 23 cases of mixed-type gastric cancer patients. Bioinformatics analysis was employed to investigate the differences in clinical characteristics and tumor microenvironment between Mes-GC and non-mesenchymal-subtype gastric cancer (non-Mes-GC) by integrating data resources including The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and National Genomics Data Center (NGDC).

Compared to non-Mes-GC patients, Mes-GC ones were characterized by later clinical stages, deeper tumor infiltration, and higher rates of lymph node metastasis. Kaplan-Meier survival analysis confirmed that Mes-GC patients were associated with shorter survival time, poor prognosis as well as increased risk of cancer recurrence (P<0.05). Single-cell RNA sequencing data revealed that tumor cells in Mes-GC showed higher expression levels of the genes related to stemness, metastasis (P<0.05), and epithelial-mesenchymal transition (EMT). And in the tumor microenvironment, there were significant more myeloid cells, smooth muscle cells, endothelial cells and fibroblasts, with the most pronounced elevation in the proportion of fibroblasts (P<0.05). Moreover, the patients with larger proportion of fibroblasts were associated with poorer prognosis.

Mes-GC tumor cells exhibit higher stemness and EMT characteristics, and stromal cells such as myeloid cells, endothelial cells, and fibroblasts are enriched in the tumor microenvironment. These features may be key factors contributing to poor prognosis and high recurrence rate of Mes-GC.

To identify the enhancer landscape marked by histone H3K27ac modifications in diffuse-type gastric cancer (DGC) tissues, and to elucidate the epigenetic remodeling mechanisms by which active enhancers regulate cachexia-related genes.

Gastric mucosal tissue samples were collected from Department of Gastroenterology of Army Medical Center of PLA during January 2022 to March 2023, including 10 normal gastric mucosa tissues (Normal group), 10 DGC tissues diagnosed with cachexia (DGC group), and 10 organoids derived from DGC tissues (Organoid group). Using H3K27ac chromatin targeting cleavage and tagmentation (CUT&Tag) technology, genomic modification regions were captured to screen specific active enhancers and their potential target genes in DGC tissues. CRISPR-dCas9 gene editing technology was used to intervene with the enhancers, and the expression of target genes was detected with Western blotting and qRT-PCR. Sixteen female SPF-grade BALB/c Nude mice (6~8 weeks old, weighing 18~21 g) were utilized to establish an orthotopic xenograft tumor model using the human diffuse-type gastric cancer cell line MKN45. Cachexia-related phenotypes were evaluated in 3 groups: normal group (n=4), silencing group (n=6), and control group (n=6).

Significant differential enhancer regions were identified between DGC and normal gastric mucosa tissues. DGC tissues exhibited a marked increase in enhancer abundance (P<0.05) and signal intensity when compared with the normal counterparts. Integrated analysis of transcriptome data revealed that some of these active enhancers up-regulated the expression of GDF15, a cachexia-associated target gene in DGC. Targeted silencing of the active enhancer of GDF15 using CRISPR/dCas9-KRAB plasmid technology resulted in a significant reduction in GDF15 expression at both mRNA levels (P<0.05) and protein. Results from orthotopic transplantation experiments of DGC demonstrated that silencing of active enhancers alleviated the cachexia phenotype in nude mice (P<0.05).

DGC exhibits enhancer remodeling, which regulates the expression of the cachexia-associated gene GDF15, and thereby contributes to the pathogenesis and progression of cancer cachexia.