miR-92a-3p在小鼠胚胎神经管闭合中的作用及机制

Effect and mechanism of miR-92a-3p on neural tube closure in mouse embryos

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 defect,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(0.753±0.052与1.006±0.126,t=3.212,P=0.033),NOX4蛋白表达高于对照组(0.870±0.039与0.688±0.056,t=4.621,P=0.010)。在转染NOX43’端非翻译区(3’untranslated regions,3’UTR)荧光素酶报告基因的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(0.368±0.102与1.000±0.149,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(1.254±0.080与1.000±0.129,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(1.077±0.142与1.432±0.300,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(1.443±0.054与1.249±0.090,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[(8.8±6.5)%与(44.1±6.8)%,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[(37.2±11.7)%与(8.8±6.5)%,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[(102.7±4.5)与(133.0±11.8)个,t=4.162,P=0.014],而共转染NOX4与miR-92a-3p后,与转染NOX4组比较,发生迁移的细胞明显增多[(176.0±11.0)与(102.7±4.5)个,t=10.680,P<0.001]。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。

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).Method C57BL/6J mice were used as 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 plasmids 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 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.Result The expression of miR-92a-3p in NTD group was lower than that in control group(0.753±0.052 vs 1.006±0.126,t=3.212,P=0.033),and NOX4 protein expression was higher than that in control group(0.870±0.039 vs 0.688±0.056,t=4.621,P=0.010).In C17.2 cells transfected with the 3'untranslated regions(3'UTR)luciferase reporter gene,the luciferase activity of co-transfected miR-92a-3p mimics was lower than that of the control group(0.368±0.102 vs 1.000±0.149,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(1.254±0.080 vs 1.000±0.129,t=2.899,P=0.044).In C17.2 cells transfected with miR-92a-3p mimic,the protein expression of NOX4 was lower than that in the mimic control group(1.077±0.142 vs 1.432±0.300,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(1.443±0.054 vs 1.249±0.090,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[(8.8±6.5)%vs(44.1±6.8)%,t=6.513,P=0.003].However,when co-transfected with NOX4 and miR-92a-3p,cell wound healing rate increased compared with NOX4 group[(37.2±11.7)%vs(8.8±6.5)%,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[(102.7±4.5)vs(133.0±11.8),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[(176.0±11.0)vs(102.7±4.5),t=10.680,P<0.001].Conclusion In mouse models of NTD,downregulated miR-92a-3p can inhibit cell migration during neural tube closure in mouse embryos through increasing the expression of NOX4,and ultimately induce NTD.