Enhancer Remodeling Characteristics Of Gastric High-grade Intraepithelial Neoplasia

Objective: With the advancement of genome sequencing,research on the mechanism of malignant tumorigenesis has become more three-dimensional.Researchers have found that enhancer,as cis-acting element,can regulate gene expression in a longrange manner,and enhancer remodeling is a common phenomenon during tumorigenesis.High-grade intraepithelial neoplasia(HGIN)has a high proportion of progression to gastric cancer,but the remodeling characteristics and regulatory mechanisms of enhancers in HGIN and gastric carcinogenesis are still unclear.Elucidating the characteristics of enhancer remodeling in HGIN and gastric carcinogenesis will provide new ideas and methods for gastric cancer prevention and personalized treatment.Methods and Results: In this study,we collected a total of 23 cases of gastric highgrade intraepithelial neoplasia and 28 cases of normal gastric mucosal tissues diagnosed under magnification endoscopy.Biopsies were taken through endoscopy and HE staining was performed to clarify the sample types without affecting the pathological diagnosis.CUT&Tag and transcript level assay were performed to characterize enhancer in the samples.The enhancer reprogramming characteristics were analyzed by comparing the H3K27 ac modification signals of high-grade intraepithelial neoplasia and normal gastric mucosa tissues.Principal component analysis(PCA)of high-grade intraepithelial neoplasia and normal gastric mucosa tissues revealed significantly different patterns of enhancer distribution between the two groups,leading to the hypothesis that enhancers play a crucial role in high-grade intraepithelial neoplasia development.Enhancer differential analysis identified 5,917 enhancer loci with increased H3K27 ac signal(HGIN-Gain VEL)and 2,841 enhancer loci with decreased H3K27 ac signal(HGIN-Loss VEL)in high-grade intraepithelial neoplasia.Genes associated with these enhancer loci were annotated using the Genomic Regions Enrichment of Annotations Tool(GREAT),and it was observed that the genes associated with enhancer gain were mainly enriched in tumorigenesis and malignant behavior-related pathways,whereas the genes associated with enhancer loss were primarily enriched in various metabolic,tight junction,and other signaling pathways.Combined with transcriptomic data analysis,the expression of classical oncogenes and stemness-related molecules associated with HGIN-Gain VEL increased with increasing levels of H3K27 ac signaling.OLFM4 showed the greatest difference in transcript levels between normal and high-grade intraepithelial neoplastic tissues.Motif analysis of all enhancer regions obtained in high-grade intraepithelial neoplasia revealed that the transcriptional level of HNF4 A was the highest.IGV visualization indicated that the transcriptional level of OLFM4 was significantly increased in high-grade intraepithelial neoplasia under the cooperative regulation of enhancers and HNF4 A.HNF4A was also highly expressed in progressive gastric cancer with poor prognosis,suggesting that it co-regulates OLFM4 with enhancers to play an important role in gastric high-grade intraepithelial neoplasia development.On the other hand,our analysis also identified a large number of genes with increased enhancer modifications but no significant increase in actual transcript levels,which we termed as primed genes.Although the primed genes were not highly expressed in high-grade intraepithelial neoplasia,they were significantly expressed in progressive cancer and enriched in tumor initiation and inflammatorycancer transformation pathways.Motif analysis of all enhancer regions regulating the primed genes identified HOXA11,a transcription factor that was not highly transcribed in high-grade intraepithelial neoplasia but significantly elevated in gastric cancer tissues.IGV visualization revealed that HOXA11 could regulate the expression of CTNNB1,which was also significantly elevated in gastric cancer tissues.We hypothesize that HOXA11 and enhancers cooperatively regulate CTNNB1 to play an important role in the progression of gastric high-grade intraepithelial neoplasia to progressive cancer.Conclusions: Our study reveals that enhancer remodeling across the genome is a crucial epigenetic feature in the development of high-grade gastric intraepithelial neoplasia,as evidenced by our epigenomic and transcriptomic integration analysis.We have identified OLFM4 as a gene that is co-regulated by enhancers and HNF4 A and consistently upregulated in high-grade intraepithelial neoplasia and progressive gastric cancer,thus promoting the development of high-grade intraepithelial neoplasia.Additionally,our analysis has uncovered that the CTNNB1 enhancer is enriched in high-grade intraepithelial neoplasia and primed for activation,and is ultimately activated by HOXA11 as an oncogene in the middle and late stages of intestinal gastric cancer development.