MicroRNA Blocks Stomach Cancer Growth by Silencing AHR and Triggering Autophagy

A small RNA molecule can rein in one of the world’s most common cancers by switching off a single gene and flipping on the cell’s self-digestion machinery, according to a new study published in Cell Division. The research, led by Qian Wan, Siwei Wang, Wei Dong, and Xinyi Liu with senior author Shanling Xu, combined large-scale analysis of The Cancer Genome Atlas stomach adenocarcinoma cohort with laboratory experiments in gastric cancer cell lines and xenograft tumors in nude mice.

The team’s starting point was a puzzle in the published literature. miR-124-3p had already been described as a tumor suppressor in liver, breast, colorectal, prostate, and bladder cancers, where its loss is tied to malignant progression. In stomach adenocarcinoma, however, its precise biological function remained insufficiently clarified. Meanwhile, the aryl hydrocarbon receptor, AHR, a ligand-activated transcription factor best known for coordinating detoxification responses, had been reported at elevated levels in gastric tumors, where it promotes proliferation and migration, inhibits apoptosis, and shapes the tumor immune microenvironment. No direct evidence had yet connected the two molecules, although the target-prediction tools TargetScan 7.1 and miRDB suggested that AHR might be a target of miR-124-3p.

Bioinformatic analysis of the TCGA-STAD dataset delivered the first hints, though not all of them pointed in the same direction. Using the LinkedOmics database across 415 stomach adenocarcinoma patients, the researchers identified significantly dysregulated microRNAs associated with AHR. Among them, miR-124-3p showed a positive correlation with AHR expression, with a Pearson correlation coefficient of 0.4267 and a p-value of 4.83 × 10−3. The authors themselves flag this tension: the positive correlation was not validated in vitro or in vivo, where an opposite, negative regulatory trend emerged, and they attribute the discrepancy to cohort composition, sequencing platforms, and normalization methods.

The clinical associations were clearer for AHR. Elevated AHR mRNA predicted worse overall survival in the TCGA cohort, with a hazard ratio of 1.46 and a p-value of 6.2 × 10−5, and Kaplan-Meier analysis linked high AHR expression to reduced survival probability with a p-value of 0.002. AHR mRNA also rose with advancing clinical stage, a pattern replicated in the independent GSE15459 validation cohort of 200 patients. As a diagnostic discriminator, AHR mRNA distinguished tumors from normal tissue with an area under the ROC curve of 0.956 in TCGA-STAD, and AUCs of 0.626 and 0.603 in the GSE15459 and GSE30070 validation cohorts. miR-124-3p, by contrast, was not a significant prognostic predictor for overall survival, with a hazard ratio of 0.75 and a p-value of 0.061, and its diagnostic AUC of 0.667 in TCGA reflected limited utility. The authors note that the validation AUC of 0.603 indicates only moderate predictive accuracy for miR-124-3p as a single biomarker.

The mechanistic core of the study came from the laboratory. In a dual-luciferase reporter assay, MKN-45 gastric cancer cells carrying the wild-type 3′-untranslated region of AHR showed a significant drop in luciferase activity when a miR-124-3p mimic was overexpressed, while a mutant construct with a disrupted binding site was unaffected. That result established a direct physical interaction between miR-124-3p and AHR mRNA. Western blotting then revealed what happens downstream: transfection with the miR-124-3p mimic markedly reduced AHR and p62 protein levels while increasing the LC3II/I ratio and Beclin-1 abundance in both MKN-45 and AGS cells, a molecular signature of autophagy induction. Chloroquine, an autophagy inhibitor, suppressed Beclin-1 and lowered the LC3II/I ratio, and it reversed the miR-124-3p-driven changes in p62 and AHR, confirming that the effect runs through the autophagy pathway.

Functional assays translated those molecular shifts into cellular behavior. Wound-healing and Transwell experiments showed that the miR-124-3p mimic markedly suppressed migration and invasion of MKN-45 and AGS cells, effects that chloroquine partially restored. Conversely, a miR-124-3p inhibitor enhanced migration and invasion. When the team knocked down Beclin-1, the essential autophagy initiator encoded by BECN1, using siRNA in both cell lines, the inhibitory effect of miR-124-3p overexpression on migration and invasion was significantly reversed, indicating that Beclin-1-mediated autophagy is a critical link in the regulatory chain. Rescue experiments completed the loop: overexpressing AHR restored the wound closure, invasion, migration, and proliferation that miR-124-3p had impaired in MKN-45 cells, demonstrating that AHR downregulation is the pivotal event.

The in vivo arm of the study extended the findings to living tumors. After four weeks, tumors in the miR-124-3p agomir group weighed significantly less than those in controls, and immunohistochemical staining showed a marked decrease in nuclear AHR-positive cells within the xenografts. The authors also report that miR-124-3p delivery mitigated cancer-induced cachexia, attenuating the body-weight loss typically seen in tumor-bearing mice. The small sample size of three mice per group, acknowledged in the paper’s limitations section, reduces statistical power, but the direction of effect was consistent with the in vitro data.

Deeper probing of the AHR pathway uncovered a second layer of regulation. Gene Ontology annotation of AHR-related processes, performed with Metascape, and protein-interaction mapping with STRING pointed toward β-catenin, the central transducer of Wnt signaling. Treating MKN-45 and AGS cells with the AHR inhibitor CH-223,191 at 0.03 micromolar, the AHR agonist ITE at 3 nanomolar, or the miR-124-3p mimic significantly reduced AHR mRNA and protein, suppressed CTNNB1 mRNA and β-catenin protein, and increased GSK-3β mRNA, while leaving c-Myc, Cyclin D1, and Wnt4 mRNA unchanged. AHR inhibition also raised the LC3II/I ratio and Beclin-1 expression, and the miR-124-3p mimic decreased phosphorylated p65 protein levels.

The study’s design, integrating bioinformatic prediction with in vitro and in vivo validation, is presented as a methodological strength, a closed loop from large-scale screening to experimental confirmation. Yet the authors are candid about the constraints. The work relied mainly on TCGA data and a limited number of clinical samples without multi-center validation, which may affect generalizability. The xenograft experiment included only three mice per group. The positive correlation between miR-124-3p and AHR seen in the LinkedOmics database, opposite to the negative regulatory relationship observed experimentally, underscores the authors’ warning that database-derived associations require functional validation before they can be trusted. They also note that gastric cancer progression involves complex molecular networks and that miR-124-3p may regulate additional pathways not explored here, while clinical application of microRNAs faces hurdles of stability, delivery efficiency, and potential off-target effects.

Looking forward, the authors propose several avenues. Larger, population-diverse clinical datasets are needed to confirm miR-124-3p’s reliability as a biomarker and to test whether it synergizes with established gastric cancer markers. Mechanistic work should identify the upstream regulators of miR-124-3p and map its role in other signaling pathways. On the therapeutic side, optimized delivery systems such as lipid nanoparticles, viral vectors, or exosome-mediated transport could improve in vivo stability and targeting, while combining CRISPR-Cas9 technology, small-molecule inhibitors, or AHR antagonists might refine AHR-targeted strategies. The authors emphasize that animal models and clinical trials will be essential to assess the safety and efficacy of any miR-124-3p-based therapy before it can move toward precision treatment of stomach adenocarcinoma. For now, the study adds a concrete molecular link, miR-124-3p to AHR to autophagy, to the growing map of regulatory circuits that gastric cancer cells must evade to progress.

Subject of Research: MicroRNA-124-3p regulation of AHR-mediated autophagy in stomach adenocarcinoma

Article Title: MiR-124-3p inhibits stomach adenocarcinoma progression by targeting AHR to induce autophagy

Article References: Wan, Q., Wang, S., Dong, W., Liu, X., Li, X., Liu, C., & Xu, S. (2025). MiR-124-3p inhibits stomach adenocarcinoma progression by targeting AHR to induce autophagy. Cell Division, 21(1), Article 6. https://doi.org/10.1186/s13008-025-00175-9

Image Credits: AI Generated

DOI: 10.1186/s13008-025-00175-9

Keywords: microRNA-124-3p, stomach adenocarcinoma, AHR, autophagy, gastric cancer, TCGA, xenograft, biomarker, tumor suppressor, beta-catenin, chloroquine, Beclin-1