Role and mechanism of Helicobacter pylori-induced enhancer RNA in the regulation of MYC to promote gastric cancer

SHI Yun; GAO Yun; YAN Caiwang; PENG Rui; LI Gang

doi:10.3969/j.issn.1674-4136.2024.05.009

Abstract：Objective This research endeavors to examine the induction of enhancer RNA(eRNA)expression by Helicobacter pylori(H.pylori)and its regulatory impact on MYC transcription.The study aims to elucidate the molecular mechanisms by which eRNA modulates MYC.Furthermore,it seeks to explore the involvement of eRNA in the onset and progression of gastric cancer.Methods Following RNA-sequencing(RNA-seq)analysis of GES-1 gastric epithelial cells co-cultured with H.pylori,the MYC gene was selectively expressed.Based on the Assay for Transposase-Accessible Chromatin with high-throughput Sequencing(ATAC-seq)and Cleavage Under Targets and Tagmentation(CUTTag)techniques,we identified the enhancer RNA(eRNA)associated with MYC,referred to as eMYC.The expression levels of eRNA and MYC were quantified before and after H.pylori infection in GES-1 cells using Reverse Transcription Quantitative Polymerase Chain Reaction(RT-qPCR).Changes in protein content were assessed by Western blot analysis.The regulatory role of eRNA on MYC expression was examined by knocking down eRNA and subsequent RT-qPCR analysis.Cleavage Under Targets and Release Using Nuclease(CUTRUN)assays were employed to explore the underlying mechanism of eRNA-mediated regulation of MYC.In the gastric cancer cell line MKN1,the changes in expression levels of eRNA and MYC after H.pylori infection were investigated.Cellular proliferation was evaluated using the 5-ethynyl-2'-deoxyuridine(EdU)incorporation assay,while apoptosis was analyzed by flow cytometric analysis.Results The oncogene MYC was upregulated following H.pylori infection(FDR＜1 × 10-300),with regions of open chromatin within 1MB upstream and downstream of MYC showing significant signals(FDR＜0.05).CUTTag results revealed high signals of H3K27ac,H3K4me1,and RNA polymerase Ⅱ in these regions.RT-qPCR assays demonstrated that both eMYC and MYC expression levels were significantly increased in GES-1 cells after H.pylori infection(P＜0.001),accompanied by an elevation in MYC protein content.Knockdown of eMYC resulted in decreased MYC expression levels(P＜0.001)and reduced protein content.CUTRUN experiments showed that upon eMYC knockdown,there was a decrease in the enrichment of H3K27ac and BRD4 at the genomic regions transcribed by eMYC(P＜0.01).In MKN1 cells,infection with H.pylori led to increased expression levels of both eMYC and MYC(P＜0.01).Knockdown of eMYC reduced cell proliferation(P＜0.001)and increased apoptosis(P＜0.05).Conclusions The H.pylori infection induces the expression of eMYC,which plays a significant role in the pathogenesis of gastric cancer.This is achieved by modulating H3K27ac methylation and facilitating the transcription of the proto-oncogene MYC through its interaction with BRD4.The elevated expression of eMYC promotes cell proliferation and inhibits apoptosis in gastric cancer cells,suggesting its significant role in the progression of gastric cancer.