Key Laboratory of Health Ministry for Congenital Malformation, Shengjing Hospital, China Medical University, Shenyang, China.
Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, China.
CNS Neurosci Ther. 2020 Sep;26(9):902-912. doi: 10.1111/cns.13383. Epub 2020 Apr 24.
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.
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.
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.
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.
神经管闭合失败导致的细胞凋亡过度会导致神经管缺陷（NTDs）。NADPH 氧化酶 4（NOX4）是细胞生长和死亡的关键介质，但它在 NTDs 中的作用尚未得到描述。NOX4 是 miR-322 的潜在靶点，我们之前的研究表明，miR-322 参与了高糖诱导的 NTDs。在这项研究中，我们研究了 NOX4 对 NTD 中胚胎神经上皮的影响，并揭示了 miR-322 的一个新的调控机制，通过改善细胞凋亡来破坏神经胚层的形成。
使用全反式视黄酸（ATRA）诱导的小鼠模型来研究 NTDs。RNA 下拉和双荧光素酶报告基因检测用于证实 NOX4 和 miR-322 之间的相互作用。在小鼠神经干细胞和全胚胎培养中，Western blot 和 TUNEL 用于研究 miR-322 和 NOX4 对 NTD 形成中神经上皮细胞凋亡的影响。
NOX4 作为 miR-322 的一个新靶点，在 ATRA 诱导的 NTD 小鼠模型中上调。在小鼠神经干细胞中，miR-322 抑制了 NOX4 的表达；进一步的，miR-322 还抑制了由 NOX4 触发的细胞凋亡。此外，在全胚胎培养中，将 miR-322 模拟物注入羊膜腔可通过沉默 NOX4 来减轻 NTD 形成中的细胞凋亡。
miR-322/NOX4 在诱导的 NTD 形成中的细胞凋亡中起关键作用，这可能为胚胎 NTDs 的机制提供新的认识，并为针对 NTDs 的潜在治疗靶点提供基础。
