摘要： 目的 探讨miR-322-5p调控烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（NOX4）的表达及改善细胞凋亡对大鼠神经管闭合的作用。方法 采用全反式维甲酸（ATRA）诱导大鼠神经管畸形（NTDs）模型。通过实时定量PCR检测miR-322-5p在ARTA诱导的大鼠胚胎脊髓中的表达情况；通过Western blotting检测NOX4及凋亡蛋白的表达情况；采用免疫组织化学技术和组织免疫荧光技术观察NOX4在胚胎神经管中的定位和表达情况；通过对C17.2神经干细胞转染miRNA-空白对照（miR-NC）、mimic-322-5p及NOX4高表达质粒，明确NOX4和miR-322-5p之间的调控作用，采用实时定量PCR检测转染后miR-322-5p的表达，并通过Western blotting检测转染后细胞NOX4以及凋亡蛋白的表达情况。结果 在ATRA诱导的大鼠NTDs模型中，NOX4表达上调，miR-322-5p表达下调。在C17.2神经干细胞中，转染miR-322-5p抑制了NOX4的表达，也抑制了凋亡蛋白的表达。结论 miR-322-5p可抑制NOX4的表达，并抑制大鼠NTDs的发生。

miR-322-5p通过NOX4调控细胞凋亡抑制大鼠神经管畸形发生

黄琬淇, 刘玉思, 顾卉, 袁正伟

中国医科大学附属盛京医院, 国家卫生健康委员会小儿先天畸形重点实验室, 沈阳 110004

收稿日期:2020-10-12出版日期:2021-07-30发布日期:2021-06-24
通讯作者:顾卉E-mail:huier.99@hotmail.com
作者简介:黄琬淇(1997-),女,硕士研究生.
基金资助:国家自然科学基金（81771595）


关键词: 神经管缺陷, miR-322-5p, 烟酰胺腺嘌呤二核苷酸磷酸氧化酶4, 细胞凋亡
Abstract: Objective To investigate the effect of miR-322-5p in regulating NOX4 expression,apoptosis,and neural tube closure in rats. Methods An all-trans-retinoic acid (ATRA)-induced rat model of neural tube defects (NTDs) was utilized to study the NTDs mitigating potential of miR-322-5p. ATRA-induced miR-322-5p expression in rat embryos was detected by real-time quantitative PCR. Expression of NOX4 and apoptotic proteins was detected by Western blotting. NOX4 localization and expression in the embryonic neural tube were observed by immunohistochemistry and immunofluorescence. A miR-negative control (miR-NC),a miR-322-5p mimic,and a NOX4 high expression plasmid were transfected into C17.2 neural stem cells to confirm regulatory interactions between NOX4 and miR-322-5p. Real-time quantitative PCR was used to detect miR-322-5p expression after transfection,and Western blotting was used to detect NOX4 and apoptotic protein expression after transfection. Results NOX4 was upregulated,and miR-322-5p was downregulated in an ATRA-induced NTDs rat model. In C17.2 neural stem cells,NOX4 was confirmed as a target of miR-322-5p,which inhibits NOX4 expression,as well as that of tested apoptotic proteins. Conclusion miR-322-5p can inhibit NOX4 expression,reducing the risk of NTDs in rats.
Key words: neural tube defect, miR-322-5p, nicotinamide adenine dinucleotide phosphate oxidase 4, apoptosis
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