Abstract: 【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). Method C57BL/6J mice were used as the test subjects and pregnant mice were randomly dividedinto NTD group and control group with 30 mice in each group. NTD model was induced by all-trans retinoicacid (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 cellC17.2 cell line. Real-time quantitative PCR (RT-qPCR) was used to detect the expression of miR-92a-3p andWestern blotting was used to detect the expression of NOX4 in embryos and C17.2 cells, respectively. The bindingand 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-wayANOVA 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, theluciferase activity of co-transfected miR-92a-3p mimics was lower than that of the control group (0.368±0.102vs 1.000±0.149, t=5.530, P=0.005). The luciferase activity of co-transfected miR-92a-3p inhibitor group washigher than that of inhibitor control group (1.254±0.080 vs 1.000±0.129, t=2.899, P=0.044). In C17.2 cellstransfected with miR-92a-3p mimic, the protein expression of NOX4 was lower than that in the mimic controlgroup (1.077±0.142 vs 1.432±0.300, t=2.396, P=0.044). On the contrary, after transfected with miR-92a-3pinhibitor, the protein expression of NOX4 was higher than that in the inhibitor control group (1.443±0.054vs 1.249±0.090, t=3.709, P=0.010). The results of cell scratch assay showed that the wound healing rate of cellstransfected 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 ofNOX4, and ultimately induce NTD.
2023, Vol. 11 
