Aims Failure of neural tube closure resulting from excessive apoptosis leads to neural tube defects (NTDs). NADPH oxidase 4 (NOX4) is a critical mediator of cell growth and death, yet its role in NTDs has never been characterized. NOX4 is a potential target of miR‐322, and we have previously demonstrated that miR‐322 was involved in high glucose‐induced NTDs. In this study, we investigated the effect of NOX4 on the embryonic neuroepithelium in NTDs and reveal a new regulatory mechanism for miR‐322 that disrupts neurulation by ameliorating cell apoptosis. Methods All‐trans‐retinoic acid (ATRA)‐induced mouse model was utilized to study NTDs. RNA pull‐down and dual‐luciferase reporter assays were used to confirm the interaction between NOX4 and miR‐322. In mouse neural stem cells and whole‐embryo culture, Western blot and TUNEL were carried out to investigate the effects of miR‐322 and NOX4 on neuroepithelium apoptosis in NTD formation. Results NOX4, as a novel target of miR‐322, was upregulated in ATRA‐induced mouse model of NTDs. In mouse neural stem cells, the expression of NOX4 was inhibited by miR‐322; still further, NOX4‐triggered apoptosis was also suppressed by miR‐322. Moreover, in whole‐embryo culture, injection of the miR‐322 mimic into the amniotic cavity attenuated cell apoptosis in NTD formation by silencing NOX4. Conclusion miR‐322/NOX4 plays a crucial role in apoptosis‐induced NTD formation, which may provide a new understanding of the mechanism of embryonic NTDs and a basis for potential therapeutic target against NTDs.

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