孕前糖尿病引起的高血糖会导致胎儿神经管缺陷（NTDs）。叶酸补充是预防NTDs的唯一有效方法，然而，部分NTDs对叶酸具有抗药性。过量的叶酸与较高的母体癌症风险和婴儿过敏有关。因此，需要额外的干预措施。了解母体糖尿病引起的NTDs的机制可以确定预防这些缺陷的潜在靶点。虽然尚未在临床上使用，但越来越多的证据表明，微小RNA（miRNAs）是胚胎发育中重要的中间产物，可以作为疾病干预的生物标志物和药物靶点。我们以往的研究表明，体内的母体糖尿病会激活在发育中胚胎中的肌醇需求性跨膜激酶/内切酶1α (IRE1α)，而体外高葡萄糖则会降低miR-322水平。IRE1α是一种RNA内切酶，然而目前尚不清楚IRE1α是否特异性地靶向和降解miR-322，或者miR-322的降解是否通过凋亡导致NTDs。我们假设IRE1α可以抑制母体糖尿病引起的NTDs中的miR-322，并且通过恢复发育中神经上皮中miR-322的表达来改善NTDs。为了确定预防母体糖尿病引起的NTDs的潜在靶点，我们研究了一个microRNA和一个RNA内切酶在暴露于母体糖尿病的小鼠胚胎中的作用和关系。为了确定miR-322的减少是否对糖尿病妊娠中的NTD形成必要，我们将携带miR-322表达转基因（miR-322 TG）的雄性小鼠与非糖尿病或糖尿病野生型（WT）雌性小鼠交配，以产生具有或没有miR-322过表达的胚胎。在胚胎尚未闭合的胚胎第8.5天（E8.5），采集胚胎进行三种miR-322转录本（初级、前体和成熟miR-322）、肿瘤坏死因子受体相关因子3（Traf3）和神经上皮细胞存活的评估。当没有NTD形成时，NTD发生率在胚胎第10.5天（E10.5）确定是否神经管已经闭合。为了确定母体糖尿病和高葡萄糖对哪种miR-322转录本起作用，对来自非糖尿病或糖尿病母鼠的胚胎以及在不同葡萄糖浓度和不同时间点处理的C17.2小鼠神经干细胞进行了三种miR-322转录本的评估。为了确定内切酶IRE1α是否靶向miR-322，使用小干扰RNA（siRNA）敲除IRE1α或通过DNA质粒转染过表达IRE1α来确定IRE1α缺陷或过表达对miR-322表达的影响。进行RNA免疫沉淀以揭示IRE1α的直接靶点。母体糖尿病抑制了发育中神经上皮中的miR-322表达。在神经上皮中恢复miR-322表达可以阻断母体糖尿病引起的caspase 3和8剪切和细胞凋亡，从而减少NTDs。通过转基因过表达逆转了由母体糖尿病抑制的miR-322在暴露于母体糖尿病的胚胎中预防了Traf3的上调。激活的IRE1α作为内切酶降解前体miR-322，导致成熟miR-322减少。本研究支持IRE1α-miR-322-Traf3回路在糖尿病妊娠中诱导神经上皮细胞凋亡和NTD形成中的重要作用，并确定IRE1α和miR-322作为预防母体糖尿病引起的NTDs的潜在靶点。版权所有 © 2023 Elsevier Inc. 保留所有权利。

Hyperglycemia from pregestational diabetes induces neural tube defects (NTDs) in the developing fetus. Folate supplementation is the only effective way for preventing NTDs, however, some cases of NTDs are resistant to folate. Excess folate has been linked to higher maternal cancer risk and infant allergy. Therefore, additional interventions are needed. Understanding the mechanisms underlying maternal diabetes-induced NTDs can identify potential targets for preventing such defects. While not yet in clinical use, growing evidence suggests that microRNAs (miRNAs) are important intermediates in embryonic development and can serve both as biomarkers and drug targets for disease intervention. Our previous studies showed that maternal diabetes in vivo activates the inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) in the developing embryo, and that high glucose in vitro reduces miR-322 levels. IRE1α is an RNA endonuclease, however it is unknown whether IRE1α targets and degrades miR-322 specifically, or whether miR-322 degradation leads to NTDs via apoptosis. We hypothesize that IRE1α can inhibit miR-322 in maternal diabetes-induced NTDs, and that restoring miR-322 expression in developing neuroepithelium ameliorates NTDs.To identify potential targets for preventing maternal diabetes-induced NTDs, we investigated the roles and relationship of a microRNA and an RNA endonuclease in mouse embryos exposed to maternal diabetes.To determine if miR-322 reduction is necessary for NTD formation in diabetic pregnancies, we mated male mice carrying a transgene expressing miR-322 (miR-322 TG) with nondiabetic or diabetic wide-type (WT) female mice to generate embryos with or without miR-322 overexpression. At embryonic day 8.5 (E8.5) when neural tube is not yet closed, embryos were harvested for the assessment of three miR-322 transcripts (primary, precursor, and mature miR-322), tumor necrosis factor receptor-associated factor 3 (Traf3), and neuroepithelium cell survival. NTD incidences were determined in E10.5 embryos when neural tube should be closed if there's no NTD formation. To identify which miR-322 transcript is affected by maternal diabetes and high glucose, three miR-322 transcripts were assessed in embryos form nondiabetic or diabetic dams and in C17.2 mouse neural stem cells treated with different concentration of glucose and at different time points. To determine if the endonuclease IRE1α targets miR-322, small interfering RNA (siRNA) knockdown of IRE1α or overexpression of IRE1α by DNA plasmid transfection were used to determine the effect of IRE1α deficiency or overexpression on miR-322 expression, respectively. RNA immunoprecipitation was performed to reveal the direct targets of IRE1α.Maternal diabetes suppressed miR-322 expression in the developing neuroepithelium. Restoring miR-322 expression in the neuroepithelium blocked maternal diabetes-induced caspase 3 and 8 cleavage and cell apoptosis, leading to an NTD reduction. Reversal of maternal diabetes-inhibited miR-322 via transgenic overexpression prevented Traf3 upregulation in embryos exposed to maternal diabetes. Activated IRE1α acted as an endonuclease and degraded precursor miR-322, resulting in mature miR-322 reduction.This study supports the critical role of the IRE1α-miR-322-Traf3 circuit in the induction of neuroepithelial cell apoptosis and NTD formation in diabetic pregnancies and identifies IRE1α and miR-322 as potential targets for preventing maternal diabetes-induced NTDs.Copyright © 2023 Elsevier Inc. All rights reserved.