Analysis of Genes Associated with Both Neural Tube Defects and Neuroectodermal Tumors

Abstract

BACKGROUND Previous studies have demonstrated that embryo development and the occurrence of tumors are closely related, as key genes, pathways, miRNAs, and other biological mechanisms are involved in both processes. Extensive research has found that abnormal development of nerve ectodermal cells not only leads to neural tube defects (NTDs), but also neuroectodermal tumors. MATERIAL AND METHODS Genes associated with both NTDs and neuroectodermal tumors were obtained from the DisGeNET database. The STRING database was used to construct the protein-protein interaction (PPI) network and the hub genes were visualized using Cytoscape. Additionally, we predicted the miRNAs targeting the identified genes. Sequencing data obtained from an NTDs mouse model and human samples were used to confirm the bioinformatics results. Moreover, a dual-luciferase report assay was used to validate the targeting relationship between the miRNA-gene pairs identified. RESULTS A total of 104 intersection genes of NTDs-related and neuroectodermal tumors-related genes were obtained; 20 of these genes were differentially expressed in NTDs samples and had very close interactions. Among 10 hub genes, we identified 3 important susceptibility genes differentially expressed both in RA-induced NTDs mice and human glioblastoma samples: Ncam1, Shh, and Ascl1. Among these, we found that the Ncam1 expression level was regulated by mmu-miR-30a-5p, and the Ascl1 expression level was regulated by mmu-miR-375-3p. CONCLUSIONS In conclusion, we identified differentially expressed genes and a potential miRNA-mediated regulation mechanism shared between NTDs and neuroectodermal tumors that may guide future studies aiming to find novel therapeutic targets for NTDs or neuroectodermal tumors.

Figure 1

The intersection genes associated with NTDs and neuroectodermal tumors. Deep pink: Genes associated with NTDs and 6 kinds tumors and their Score_gda were higher than the average; Light pink: Genes associated with NTDs only and their Score_gda were less than the average; Orange: Genes associated with 6 kinds of tumors only and their Score_gda were higher than the average; Blue: Genes associated with 6 kinds of tumors only and their Score_gda were less than the average. This figure was created using PowerPoint, version Microsoft Office Home and Student 2019, supported by Microsoft.

Figure 2

Expression of 27 differentially expressed gene in E8.5, E9.5, and E10.5. (A) Heatmap of DGEs’ expression; (B–D) X-axis: Log2 Ratio in E8.5 (RA/Con), Log2 Ratio in E9.5 (RA/Con), Log2 Ratio in E10.5 (RA/Con) respectively, Y-axis: Gene Symbol. Flesh colored: genes with |log2 Ratio|>1 and P value<0.05; light gray: genes with |log2 Ratio|<1 or P value>0.05. These figures were created by an open-source tool RStudio, version 1.1.456.

Figure 3

Interactive network of 20 differentially expressed genes. (A) The protein–protein interaction (PPI) network of the DEGs. (B) Hub genes. The PPI network of the DEGs was established through STRING (Search Tool for the Retrieval of Interacting Genes database) website, visualized by Cytoscape, and the hub genes were explored using the CytoHubba plug-in, version 3.42, which is an open-source software platform.

Figure 4

Differential expression of miRNA-mRNA pairs in E9.5 and E10.5. (A) mmu-miR-30a-5p-Ncam1; (B) mmu-miR-375-3p-Ascl1; (C) mmu-miR-9-5p-Cdh1; Gray: Control; Red: Upregulated in NTDs; Green: Downregulated in NTDs. n=3,* P<0.05, ** P<0.01, *** P<0.001. Data were analyzed and visualized using the software GraphPad Prism, version 8.0, supported by GraphPad Software.

Figure 5

The expression of candidate genes in E9.5. Gray: Control; Red: Upregulated in NTDs; Green: Downregulated in NTDs. n=3,* P<0.05, ** P<0.01, *** P<0.001. Data were analyzed and visualized using the software GraphPad Prism, version 8.0, supported by GraphPad Software.

Figure 6

The expression of candidate genes in E10.5. Gray: Control; Red: Up-regulated in NTDs; Green: Downregulated in NTDs. n=3,* P<0.05, ** P<0.01, *** P<0.001. Data were analyzed and visualized using GraphPad Prism, version 8.0, supported by GraphPad Software.

Figure 7

The expression of candidate genes in GBM samples. (A) NCAM1. (B) CDH1. (C) SHH. (D) ASCL1. n=10,* P<0.05, ** P<0.01, *** P<0.001, ns – not significant. Data were analyzed and visualized using GraphPad Prism, version 8.0, supported by GraphPad Software.

Figure 8

Cell transfection efficiency. (A) Before transfection; (B) After transfection. The figure was created using Olympus CKX53-FL manufactured by Olympus Corporation.

Figure 9

Interaction between selected miRNA-mRNA pairs identified by luciferase reporter assays. (A) Luciferase assay of mmu-miR-30a-5p-Ncam1. (B) Luciferase assay of mmu-miR-375-3p-Ascl1. (C) Luciferase assay of mmu-miR-9-5p-Cdh1. **** P<0.0001. Data were analyzed and visualized using the software GraphPad Prism, version 8.0, supported by GraphPad Software.

Figure 10

Sequence alignments of miRNAs and target mRNAs. (A) mmu-miR-30a-5p and Ncam1. (B) mmu-miR-375-3p and Ascl1. (C) mmu-miR-9-5p and Cdh1. Predicted by StarBase V2.0, which is an open-source software platform. Visualized by PowerPoint, version Microsoft Office Home and Student 2019, supported by Microsoft.