Essential role of ALKBH5-mediated RNA demethylation modification in bile acid-induced gastric intestinal metaplasia. Essential role of ALKBH5-mediated RNA demethylation modification in bile acid-induced gastric intestinal metaplasia

Reflux of bile acids and subsequent caudal-related homeobox 2 (CDX2) activation contribute to gastric intestinal metaplasia (IM), which is a precursor of gastric cancer. However, the underlying mechanism by which bile acids cause this is not entirely clear. Here we demonstrated that alkylation repair homolog protein 5 (ALKBH5), which is a major RNA N6-adenosine demethylase, was required for bile acid-induced gastric IM. Mechanistically, we revealed the N6-methyladenosine (m6A) modification profile in gastric IM for the first time and identified ZNF333 as a bona fide m6A target of ALKBH5. ALKBH5 was shown to demethylate ZNF333 mRNA, leading to enhanced ZNF333 expression by abolishing m6A-YTHDF2-dependent mRNA degradation. In addition, ALKBH5 activated CDX2 and downstream intestinal markers by targeting the ZNF333/CYLD axis and activating NF-κB signaling. Reciprocally, p65, which is the key transcription factor of the canonical NF-κB pathway, enhanced the transcription activity of ALKBH5 in the nucleus, thus forming a positive feed-forward circuit. In addition, ALKBH5 levels were positively correlated with ZNF333 and CDX2 in IM tissues, indicating a significant clinical relevance. Collectively, our findings suggest an m6A modification-associated positive feed-forward loop between ALKBH5 and NF-κB signaling is involved in the generation of the IM phenotype from gastric epithelial cells. Targeting the ALKBH5/ZNF333/CYLD/CDX2 axis might be a useful therapeutic strategy for gastric IM in patients with bile regurgitation. Overall design: We compared the RNA m6A sites in CDCA treated GES-1 cells and control, and input was analyzed for gene expression level.

胆汁酸反流及随后尾侧型同源盒转录因子2（caudal-related homeobox 2, CDX2）的激活，参与了胃黏膜肠上皮化生（gastric intestinal metaplasia, IM）的发生过程——而IM正是胃癌的癌前病变。然而，胆汁酸诱发该病变的具体分子机制尚未完全阐明。本研究证实，作为主要的RNA N6-腺苷酸去甲基化酶，烷基化修复同源蛋白5（alkylation repair homolog protein 5, ALKBH5）在胆汁酸诱导的胃黏膜肠上皮化生过程中发挥了必需作用。机制上，我们首次揭示了胃黏膜肠上皮化生中的N6-甲基腺苷（N6-methyladenosine, m6A）修饰谱，并鉴定出ZNF333为ALKBH5的确证性m6A靶基因。实验表明，ALKBH5可对ZNF333 mRNA进行去甲基化修饰，通过消除m6A-YTHDF2依赖的mRNA降解通路，提升ZNF333的表达水平。此外，ALKBH5可通过靶向ZNF333/CYLD轴并激活NF-κB信号通路，进而激活CDX2及其下游肠上皮标志物的表达。反之，经典NF-κB通路的关键转录因子p65可在细胞核内增强ALKBH5的转录活性，由此形成正向前馈环路。进一步研究发现，IM组织中ALKBH5的表达水平与ZNF333及CDX2的表达呈正相关，提示该通路具有显著的临床相关性。综上，本研究结果表明，ALKBH5与NF-κB信号通路之间存在m6A修饰介导的正向前馈环路，该环路参与了胃上皮细胞向肠上皮化生表型的转化过程。靶向ALKBH5/ZNF333/CYLD/CDX2轴或许可为胆汁反流相关胃黏膜肠上皮化生患者提供潜在的治疗策略。整体实验设计：本研究对经鹅脱氧胆酸（chenodeoxycholic acid, CDCA）处理的GES-1细胞与对照组细胞的RNA m6A结合位点进行了对比分析，并通过input对照样本测序检测基因表达水平。