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

ABSTRACT: Bile acid reflux and subsequent caudal-related homeobox 2 (CDX2) activation contribute to gastric intestinal metaplasia (IM), a precursor of gastric cancer; however, the mechanism underlying this phenomenon is unclear. Here, we demonstrate that alkylation repair homolog protein 5 (ALKBH5), a major RNA N⁶-adenosine demethylase, is 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 novel 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, the key transcription factor of the canonical NF-κB pathway, enhanced the transcription activity of ALKBH5 in the nucleus, thus forming a positive feedforward circuit. Furthermore, ALKBH5 levels were positively correlated with ZNF333 and CDX2 levels in IM tissues, indicating significant clinical relevance. Collectively, our findings suggest that an m⁶A modification-associated positive feedforward loop between ALKBH5 and NF-κB signaling is involved in generating the IM phenotype of gastric epithelial cells. Targeting the ALKBH5/ZNF333/CYLD/CDX2 axis may be a useful therapeutic strategy for gastric IM in patients with bile regurgitation.

SUBMITTER: Yue B

PROVIDER: S-EPMC8479281 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Intestinal metaplasia (IM) is a premalignant lesion associated with gastric cancer. Both animal and clinical studies have revealed that bile acid reflux and subsequent chronic inflammation are key causal factors of IM. Previous studies indicated that SOX2, the key transcription factor in gastric differentiation, was downregulated during IM development while CDX2, the pivotal intestine-specific transcription factor was upregulated significantly. However, it remains unclear whether the downregulation of SOX2 promotes gastric IM emergence or is merely a concomitant phenomenon. In addition, the underlying mechanisms of SOX2 downregulation during IM development are unclear. Methods:Gastric cell lines were treated with deoxycholic acid (DCA) in a dose-dependent manner. The expression of CDX2 and miR-21 in gastric tissue microarray were detected by immunohistochemistry and in situ hybridization. Coimmunoprecipitation and immunofluorescence were performed to ascertain the interaction of SOX2 and CDX2. Luciferase reporter assays were used to detect the transcriptional activity of CDX2, and confirm miR-21 binding to SOX2 3'-UTR. The protein level of SOX2, CDX2 and downstream IM-specific genes were investigated using western blotting. mRNA level of miR-21, SOX2, CDX2 and downstream IM-specific genes were detected by qRT-PCR. Results:Bile acid treatment could suppress SOX2 expression and simultaneously induce expression of CDX2 in gastric cell lines. Furthermore, we demonstrated that SOX2 overexpression could significantly inhibit bile acid- and exogenous CDX2-induced IM-specific gene expression, including KLF4, cadherin 17 and HNF4? expression. In contrast, SOX2 knockdown had the opposite effect. A dual-luciferase reporter assay demonstrated that SOX2 overexpression could significantly suppress CDX2 transcriptional activity in HEK293T cells. CDX2 and SOX2 could form protein complexes in the nucleus. In addition, bile acid induced the expression of miR-21. The inhibition of SOX2 in bile acid-treated gastric cell lines was rescued by miR-21 knockdown. Conclusions:These findings suggested that SOX2 can interfere with the transcriptional activity of CDX2 in bile acid-induced IM and that miR-21 might play a key role in this process, which shed new lights in the prevention of gastric cancer.

Project description:Intestinal metaplasia (IM) is the inevitable precancerous stage to develop intestinal-type gastric cancer (GC). Deoxycholic acid (DCA) is the main bile acid (BA) component of duodenogastric reflux and has shown an increased concentration during the transition from chronic gastritis to IM associated with continued STAT3 activation. However, the mechanisms underlying how DCA facilitates IM in the gastric epithelium need exploration. We evaluated IM and bile reflux in corpus tissues from 161 subjects undergoing GC screening. Cell survival and proliferation, proinflammatory cytokine expression and TGR5/STAT3/KLF5 axis activity were measured in normal human gastric cells, cancer cells, and organoid lines derived from C57BL/6, FVB/N and insulin-gastrin (INS-GAS) mice treated with DCA. The effects of DCA on IM development were determined in INS-GAS mice with long-term DCA supplementation, after which the gastric bacterial and BA metabolic profiles were measured by 16S rRNA gene sequencing and LC-MS. We revealed a BA-triggered TGR5/STAT3/KLF5 pathway in human gastric IM tissues. In gastric epithelial cells, DCA promoted proliferation and apoptotic resistance, upregulated proinflammatory cytokines and IM markers, and facilitated STAT3 phosphorylation, nuclear accumulation and DNA binding to the KLF5 promoter. DCA triggered STAT3 signaling and the downstream IM marker KLF5 in mouse gastric organoids in vitro and in vivo. In INS-GAS mice, DCA promoted the accumulation of serum total BAs and accelerated the stepwise development of gastric IM and dysplasia. DCA induced gastric environmental alterations involving abnormal BA metabolism and microbial dysbiosis, in which the Gemmobacter and Lactobacillus genera were specifically enriched. Lactobacillus genus enrichment was positively correlated with increased levels of GCA, CA, T-α-MCA, TCA and β-MCA in DCA-administrated INS-GAS mice. DCA promotes nuclear STAT3 phosphorylation, which mediates KLF5 upregulation associated with gastric inflammation and IM development. DCA disturbs the gastric microbiome and BA metabolism homeostasis during IM induction.

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

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