Supplementary Material for: Upregulation of miRNA-26a enhances the apoptosis of cerebral neurons by targeting EphA2 and inhibiting the MAPK pathway

Neural tube defects (NTDs) constitute the second most common congenital malformation of the central nervous system. The pathogenesis of NTDs is not entirely clear. In recent years, microRNAs have become a hot spot in genetic and developmental biology research. The present study aimed to explore the potential role of microRNA (miRNA)-26a in NTDs and the underlying pathogenesis thereof. First, we found significantly increased miRNA-26a expression in fetuses with NTDs (p<0.0001), which significantly downregulated EphA2 and ERK1 mRNA and protein expression levels in fetuses with NTDs compared to normal controls (p<0.01). In addition, dual-luciferase reporter assay showed that miR-26a negatively regulated EphA2 by directly binding with the 3'-untranslated region of EphA2. Second, upregulation of miRNA-26a expression increased caspase 3 and 9 protein expression levels (p<0.01); and decreased EphA2 mRNA and protein expression levels (p<0.01), as well as ERK1 and SRF protein expression levels (p<0.01) in mouse neural stem cells (NE-4C) and human astroblastoma cells (U87MG). Furthermore, the upregulation of miRNA-26a inhibited cell proliferation and enhanced apoptosis of NE-4C and U87MG cells (p<0.05). Similar results were observed with the MAPK inhibitor PD98059 (p<0.01). These results suggest that miR-26a downregulates EphA2 to regulate SRF through the MAPK pathway, which affects nervous system development miR-26a targets EphA2, modulates phosphorylation of MAPK/ERK(MEK) pathway, regulates SRF, and participates in regulating nervous cell proliferation and apoptosis. Dysregulation of the aforementioned mechanism may be involved in the pathogenesis of NTDs.