1、发育医学电子杂志 2023 年 7 月 第 11 卷 第 4 期 J Dev Med(Electronic Version),Jul.2023,Vol.11,No.4241miR-92a-3p 在小鼠胚胎神经管闭合中的作用及机制张燕秋付佳琳黄琬淇袁正伟顾卉（中国医科大学附属盛京医院卫生部小儿先天畸形重点实验室，辽宁沈阳110004）论著【摘要】目的 探讨 miR-92a-3p 与烟酰胺腺嘌呤二核苷酸磷酸氧化酶 4（nicotinamide adenine dinucleotide phosphate oxidase 4，NOX4）之间的相互作用，以及两者在神经管畸形（neural tube d
2、efect，NTD）中对细胞迁移的影响。方法实验动物采用 C57BL/6J 小鼠，孕鼠随机分为 NTD 组与对照组，每组 30 只，NTD 组采用全反式维 A 酸（all-trans retinoic acid，ATRA）诱导 NTD 模型，于 E9.5 采集胚胎样本。实验所用细胞系为小鼠神经干细胞 C17.2，转染 NOX4 过表达质粒、miR-92a-3p 模拟物/抑制剂、模拟物对照/抑制剂对照。采用实时定量 PCR（real-time quantitative PCR，RT-qPCR）检测胚胎和C17.2 细胞中 miR-92a-3p 表达，采用蛋白质印迹法检测 NOX4 的表达。通过双
3、荧光素酶报告基因实验明确 miR-92a-3p 对 NOX4 的结合及靶向调控关系。采用细胞划痕实验与 Transwell 实验观察 miR-92a-3p 和NOX4 对细胞迁移活动的影响。统计学方法采用单因素方差分析、独立样本 t 检验。结果NTD 组胚胎 miR-92a-3p 表达低于对照组（0.7530.052 与 1.0060.126，t=3.212，P=0.033），NOX4 蛋白表达高于对照组（0.8700.039 与 0.6880.056，t=4.621，P=0.010）。在转染 NOX4 3 端非翻译区（3 untranslated regions，3 UTR）荧光素酶报告基因
4、的 C17.2 细胞中，共转染 miR-92a-3p 模拟物组的荧光素酶活性低于模拟物对照组（0.3680.102 与 1.0000.149，t=5.530，P=0.005）；共转染 miR-92a-3p 抑制剂组的荧光素酶活性高于抑制剂对照组（1.2540.080 与 1.0000.129，t=2.899，P=0.044）。C17.2 细胞转染 miR-92a-3p 模拟物组，NOX4 的蛋白表达低于模拟物对照组（1.0770.142 与 1.4320.300，t=2.396，P=0.044）；转染 miR-92a-3p 抑制剂组，NOX4 的蛋白表达高于抑制剂对照组（1.4430.054
5、与 1.2490.090，t=3.709，P=0.010）。细胞划痕的实验结果表明，转染 NOX4 质粒后，细胞伤口愈合的速度低于对照组 （8.86.5）%与（44.16.8）%，t=6.513，P=0.003，然而当过表达 NOX4 的同时转染 miR-92a-3p，与转染 NOX4 组比较，细胞伤口愈合速度加快 （37.211.7）%与（8.86.5）%，t=3.680，P=0.021。Transwell 实验发现，转染 NOX4 质粒后，迁移的细胞数量低于对照组 （102.74.5）与（133.011.8）个，t=4.162，P=0.014，而共转染 NOX4 与 miR-92a-3p
6、后，与转染 NOX4 组比较，发生迁移的细胞明显增多（176.011.0）与（102.74.5）个，t=10.680，P0.001。结论小鼠 NTD 模型中，异常低表达的 miR-92a-3p 能够通过上调 NOX4 的表达，阻碍小鼠胚胎神经管闭合过程中细胞的迁移活动，最终导致 NTD的发生。【关键词】神经管畸形；细胞迁移；miR-92a-3p；烟酰胺腺嘌呤二核苷酸磷酸氧化酶 4Effect and mechanism of miR-92a-3p on neural tube closure in mouse embryosZhang Yanqiu,Fu Jialin,Huang Wanqi,
7、Yuan Zhengwei,Gu Hui(Key Laboratory of Health Ministry for Congenital Malformation,Shengjing Hospital of China Medical University,Liaoning,Shenyang 110004,China)DOI：10.3969/j.issn.2095-5340.2023.04.001基金项目：国家自然科学基金（81771595；82271730）通信作者：顾卉（Email：）242发育医学电子杂志 2023 年 7 月 第 11 卷 第 4 期 J Dev Med(Electr
8、onic Version),Jul.2023,Vol.11,No.4Corresponding author:Gu Hui (Email:)【Abstract】Objective To investigate the interaction of miR-92a-3p and nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4),and the impact of them on cell migration in neural tube defect(NTD).MethodC57BL/6J mice were used as
9、the test subjects and pregnant mice were randomly divided into NTD group and control group with 30 mice in each group.NTD model was induced by all-trans retinoic acid(ATRA).Embryo samples were collected at E9.5.The miR-92a-3p mimic/inhibitor,mimic control/inhibitor control,and NOX4 high-expression p
10、lasmids were transfected into the mouse neural stem cell C17.2 cell line.Real-time quantitative PCR(RT-qPCR)was used to detect the expression of miR-92a-3p and Western blotting was used to detect the expression of NOX4 in embryos and C17.2 cells,respectively.The binding and targeting relationship of
11、 miR-92a-3p to NOX4 was clarified by double luciferase.Finally,the effect of miR-92a-3p and NOX4 on cell migration activity was observed using cell scratch assay and Transwell assay.One-way ANOVA and independent sample t-test were used for statistical analysis.ResultThe expression of miR-92a-3p in N
12、TD group was lower than that in control group(0.7530.052 vs 1.0060.126,t=3.212,P=0.033),and NOX4 protein expression was higher than that in control group(0.8700.039 vs 0.6880.056,t=4.621,P=0.010).In C17.2 cells transfected with the 3 untranslated regions(3 UTR)luciferase reporter gene,the luciferase
13、 activity of co-transfected miR-92a-3p mimics was lower than that of the control group(0.3680.102 vs 1.0000.149,t=5.530,P=0.005).The luciferase activity of co-transfected miR-92a-3p inhibitor group was higher than that of inhibitor control group(1.2540.080 vs 1.0000.129,t=2.899,P=0.044).In C17.2 cel
14、ls transfected with miR-92a-3p mimic,the protein expression of NOX4 was lower than that in the mimic control group(1.0770.142 vs 1.4320.300,t=2.396,P=0.044).On the contrary,after transfected with miR-92a-3p inhibitor,the protein expression of NOX4 was higher than that in the inhibitor control group(
15、1.4430.054 vs 1.2490.090,t=3.709,P=0.010).The results of cell scratch assay showed that the wound healing rate of cells transfected with NOX4 plasmid was lower than that of the control group(8.86.5)%vs(44.16.8)%,t=6.513,P=0.003.However,when co-transfected with NOX4 and miR-92a-3p,cell wound healing
16、rate increased compared with NOX4 group(37.211.7)%vs(8.86.5)%,t=3.680,P=0.021.Transwell assay found that the number of migrating cells transfected with NOX4 plasmid was less than that of the control group(102.74.5)vs(133.011.8),t=4.162,P=0.014.However,after co-transfected with NOX4 and miR-92a-3p,the number of migrating cells increased significantly compared with NOX4 group(176.011.0)vs(102.74.5),t=10.680,P0.001.Conclusion In mouse models of NTD,downregulated miR-92a-3p can inhibit cell migratio