Objective To map the genome-wide distribution profile of histone H3K27me3 modification in diffuse gastric cancer tissues,identify target genes regulated by H3K27me3,and primarily explore the potential mechanism of its modification reprogramming in the occurrence and development of the tumor.Methods Normal gastric mucosal tissues and diffuse gastric cancer tissues were harvested from the patients who underwent examinations or treatments in the departments of gastroenterology and gastrointestinal surgery of our medical center between 2021 and 2023.There were 14 patients in the normal group(6 males and 8 females,average age of 46 years)and 14 patients in the gastric cancer group(8 males and 6 females,average age of 63 years).Cleavage under target and tagmentation(CUT&Tag)technology was employed to capture genomic regions modified by H3K27me3,and analyze the reprogramming characteristics of these modifications.RNA sequencing data,data from high-throughput chromosome conformation capture(Hi-C)technology,and publicly available single-cell data were integrated to investigate the target genes regulated by the reprogramming of H3K27me3 modifications in diffuse gastric cancer cells.Results The quality of the CUT&Tag and RNA sequencing data met the standards required for subsequent analysis.Histone H3K27me3 modifications in normal gastric mucosa and diffuse gastric cancer tissues were primarily distributed in distal intergenic regions and intronic regions.In gastric cancer tissues,compared to normal tissues,there was significant reprogramming of H3K27me3 modifications,characterized by a marked reduction in overall H3K27me3 signal intensity.The loss of 2 912 H3K27me3 signal peaks might lead to the up-regulation of 822 tumor-associated genes.Among them,56 genes displayed the most significant up-regulation(fold change in signal intensity≥2,P<0.05),with notable enrichment in the mammalian target of rapamycin complex 1(mTORC1)signaling pathway.Specifically,the methionine transporter SLC7A5 and the cystine transporter SLC7A11 were found to have the highest expression levels in gastric cancer tissues.Single-cell data revealed that the abnormal overexpression of SLC7A11 in diffuse gastric cancer was primarily observed in tumor epithelial cells.Further validation using public data and immunohistochemical experiments confirmed the elevated expression of SLC7A11 in diffuse gastric cancer,which is associated with poor prognosis in gastric cancer patients.Conclusion The reprogramming of histone H3K27me3 modification is an important epigenetic characteristic in diffuse gastric cancer.Loss of H3K27me3 signal peaks may up-regulate the expression of SLC7A11 in diffuse gastric cancer cells,and thereby promote tumor progression.

Objective 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.Methods 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.Results 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 6 257 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).Conclusion There is the remodeling of H3K27me3 silencers in SRCC,and EHF may potentially play a crucial role in the SRCC malignant progression.

Background:The core circadian clock gene Bmal1 has been shown to be involved in the formation of visceral sensitization in irritable bowel syndrome(IBS)by affecting the tryptophan hydroxylase 1(TPH1)-5-hydroxytryptamine(5-HT)pathway,but the exact mechanism of its regulation is unknown.Aims:To investigate the molecular mechanism by which Bmal1 regulates the TPH1-5-HT pathway through the clock-controlled gene Piezo1.Methods:Dexamethasone was used to synchronize the expression of the circadian clock genes in RIN-14B cells.Bmal1 expression was up-regulated or down-regulated in RIN-14B cells by plasmid and siRNA transfection of the enterochromaffin cell(EC)model.The expression levels of target genes and proteins were detected by immunofluorescence staining,RT-qPCR,and Western blotting.5-HT content was detected by ELISA method.Results:(1)This study was the first to report the oscillation characteristics of RIN-14B circadian clock genes in EC model,among which the oscillation of Bmal1 was the most significant.Immunofluorescence showed that RIN-14B cells expressed CGA,Bmal1 and Piezo1.(2)After transfected with the Bmal1 overexpression plasmid,the mRNA and protein expression of Bmal1 were significantly up-regulated in RIN-14B cells compared with the negative control group(all P<0.001);while transfected with Bmal1 siRNA significantly decreased the mRNA and protein expression of Bmal1 in RIN-14B cells compared with the negative control group(all P<0.05).(3)After transfected with Bmal1 overexpression plasmid,the mRNA and protein expression of Piezo1,the protein expression of TPH1,and the intracellular content of 5-HT were significantly increased(all P<0.051).(4)After transfected with Bmal siRNA,mRNA expression of Piezo1 and TPH1 in RIN-14B cells was significantly down-regulated(all P<0.05),and the protein expression of Piezo1,TPH1,the intracellular 5-HT content tended to be decreased by 21%,31%,and 10%,respectively.Conclusions:RIN-14B cells have the characteristics of rhythmic oscillation of circadian clock genes,and Bmal1 overexpression and underexpression EC models can be successfully established by using RIN-14B cells.Overexpression and underexpression of Bmal1 can lead to significant changes in the clock-controlled Piezo1-TPH1-5-HT signaling pathway,suggesting that Bmal1 can be expressed by clock-control signals through the Piezo1-TPH1 pathway.This suggests that Bmal1 may be involved in the development of visceral hypersensitivity in IBS through regulation of 5-HT synthesis by the clock-controlled gene Piezo1.

Background:The core circadian clock gene Bmal1 has been shown to be involved in the formation of visceral sensitization in irritable bowel syndrome(IBS)by affecting the tryptophan hydroxylase 1(TPH1)-5-hydroxytryptamine(5-HT)pathway,but the exact mechanism of its regulation is unknown.Aims:To investigate the molecular mechanism by which Bmal1 regulates the TPH1-5-HT pathway through the clock-controlled gene Piezo1.Methods:Dexamethasone was used to synchronize the expression of the circadian clock genes in RIN-14B cells.Bmal1 expression was up-regulated or down-regulated in RIN-14B cells by plasmid and siRNA transfection of the enterochromaffin cell(EC)model.The expression levels of target genes and proteins were detected by immunofluorescence staining,RT-qPCR,and Western blotting.5-HT content was detected by ELISA method.Results:(1)This study was the first to report the oscillation characteristics of RIN-14B circadian clock genes in EC model,among which the oscillation of Bmal1 was the most significant.Immunofluorescence showed that RIN-14B cells expressed CGA,Bmal1 and Piezo1.(2)After transfected with the Bmal1 overexpression plasmid,the mRNA and protein expression of Bmal1 were significantly up-regulated in RIN-14B cells compared with the negative control group(all P<0.001);while transfected with Bmal1 siRNA significantly decreased the mRNA and protein expression of Bmal1 in RIN-14B cells compared with the negative control group(all P<0.05).(3)After transfected with Bmal1 overexpression plasmid,the mRNA and protein expression of Piezo1,the protein expression of TPH1,and the intracellular content of 5-HT were significantly increased(all P<0.051).(4)After transfected with Bmal siRNA,mRNA expression of Piezo1 and TPH1 in RIN-14B cells was significantly down-regulated(all P<0.05),and the protein expression of Piezo1,TPH1,the intracellular 5-HT content tended to be decreased by 21%,31%,and 10%,respectively.Conclusions:RIN-14B cells have the characteristics of rhythmic oscillation of circadian clock genes,and Bmal1 overexpression and underexpression EC models can be successfully established by using RIN-14B cells.Overexpression and underexpression of Bmal1 can lead to significant changes in the clock-controlled Piezo1-TPH1-5-HT signaling pathway,suggesting that Bmal1 can be expressed by clock-control signals through the Piezo1-TPH1 pathway.This suggests that Bmal1 may be involved in the development of visceral hypersensitivity in IBS through regulation of 5-HT synthesis by the clock-controlled gene Piezo1.