Construction and analysis of lncRNA-related ceRNA network in mouse model of methotrexate-induced neural tube defect under folate deficiency

Abstract

Objective To construct a regulatory network of long noncoding RNA (lncRNA)-related competing endogenous RNAs (ceRNA) in low folate combined with methotrexate (MTX) induced neural tube defects (NTDs) mouse model, and to explore the potential lncRNAs regulation in low folate-induced NTDs. Methods Forty SPF-grade 7~8-week-old healthy female C57BL/6J mice (body weight 18~22 g) and 20 healthy male C57BL/6J mice (20~22 g) were selected to this experiment. After a folate deficiency induced fetal mouse NTD model was successfully established, the embryonic brain tissues were extracted for total RNA and RNA high-throughput sequencing (RNA-seq) was performed. The results of RNA-seq were used to carry out co-expression network construction, GO function enrichment of mRNA in co-expression network and KEGG pathway enrichment analysis, construction of lncRNA-related ceRNA regulation network and other bioinformatics analysis. And the relative expression of lncRNA Xist, Sema4f and Padi2 in the fetal mouse and cell samples was verified by qPCR. Results A total of 47 differential lncRNAs (Log2FC >0.585 or Log2FC < -0.585, FDR < 0.05) were detected by RNA-seq, of which 38 were up-regulated and 9 were down-regulated. The co-expression network between lncRNA and mRNA was constructed. The results of KEGG/GO analysis on mRNAs in the co-expression network indicated that tube development (P < 0.001), collagen metabolic process (P=0.002), system development (P=0.002) and other significant enrichments were highly relevant to NTDs. Besides, KEGG pathway analysis displayed that the PI3K-Akt pathway was closely related to NTDs; lncRNA Xist, LOC102633899, 1700086L19Rik and other targeting differential genes Sema4f, Padi2, Lin28a were found in the lncRNA-related ceRNA network. qPCR confirmed that the above results in the fetal mice and cell samples under folic acid deficiency, with consistent corresponding sequences, and obviously down-regulated (P < 0.05). Conclusion In folic acid deficiency-induced mouse NTD model, lncRNA Xist, and its targeting genes Sema4f and Padi2 are obviously down-regulated.

Keywords

• neural tube defects
• epigenetics
• long noncoding rna
• competing endogenous rna