Impact Of Anti-folate Drug MTX On The Development Of Neural Tube Defects In Mice And Genomic Instability

Objects1. To establish neural tube defects (NTDs) mouse model using anti-folate drug MTX.2. To detect genomic DNA copy number variation (CNV) and DNA methylation alteration of NTD embryonic nervous tissues induced by MTX.3. To observe Neuroepithelial cell proliferation and apoptosis of neural tube defects mice induced by MTX.Methods1. Establishment of NTD mouse model using anti-folate drug MTX.Pregnant mice were treated with different doses of MTX by intraperitoneal injection on gestational day 7.5, and sacrificed on gestational day 11.5; The embryos were carefully checked and the malformation rates were recorded; Body weight, height were measured; Some embryos were fixed in cold 10% neutral buffered formalin over night, dehydrated in graded solutions of alcohol, cleared in xylene, and paraffin embedded. Sections were sequentially cut at 5 mm increments and stained with hematoxylin and eosin. Optimum dose were selected according to the results.2. Detection genomic DNA copy number variation(CNV) of NTD embryonic nervous tissues induced by MTX using Array comparative genomic hybridization(aCGH) CNV s were detected on genomic DNA from normal and NTD embryos using an array CGH chip; Based on criteria of the log2 ratio≥0.45 and segment size ? 10 kb, consecutive probes in a segment≥5, 3CNVs on chromosome X were high confidence and confirmed by the results of RT-PCR and MassARRAY platform; Gene Ontology (GO) was applied to the annotation of genes located on the 3 CNVs.3. Analysis of DNA methylation landscape around promoter CpG islands of genomic DNA from normal and NTD embryonic nervous tissues using MeDIP-chip. DNA methylation landscape around promoter CpG islands of genomic DNA from normal and NTD embryos was analyzed using MeDIP-chip. Based on criteria of peak found on all 3 NTD samples compared to normal sample, log2 ratio means?0.5, 30 peaks on chromosome X were high confidence; Gene Ontology (GO) was applied to the annotation of genes regulated by these peaks; mRNA expression was observed by RT-PCR.4. Observation on neuroepithelial cell proliferation and apoptosis of neural tube defects mice induced by MTX.TUNEL was used to observe neuroepithelial cell apoptosis of normal and NTD embryos; Immunohistochemisty method was detect expression of pH3 protein on neuroepithelial cell, which reflecting cellular proliferation; Expression of caspase3 protein on embryonic brain using immunohistochemisty and western-blotting.Results1. We successfully developed NTD model by intraperitoneal injection of MTX 4.5mg/kg on gestational day 7.5 with NTDs rate of 31.4%; The DHFR activities decreased within 4-8 hours and started to recover at 12 hours but were still lower than that before injection; Results of HE staining showed that most of NTDs were anencephaly, exhibiting lesions extending into the hindbrain (holoanencephaly), other NTDs were anencephaly and craniorachischisis.2. Compared with normal samples, 9 CNVs on chromosome 6 showing a relative amplification of NTD samples and varied in size from 2 kb to 5 kb, and 12 CNVs on chromosomes 7, 9, 11, and X showing a relative deletion of NTD samples and varied in size from 5 kb to 18.8 Mb(p <0.05). We defined 3 high-confidence CNVs on chromosome X based on criteria of the log2 ratio shift≥0.45, segment length ?10kb, consecutive probes in a segment≥5; The high-confidence CNVs were confirmed by RT-PCR and MassARRAY; GO result showed that 25/103 genes located on the 3 CNVs participate in cellular metabolic process, the other susceptibility genes are enriched signaling, developmental process, apoptosis, cell cycle, and cell communication among other annotations.3. Compared with normal samples, 35 regions showing hypomethylation and 97 regions showing hypermethylation of NTD samples, 26 region on chromosome X was high confidence based on criteria of peak found on all 3 NTD samples compared to normal sample, log2 ratio means≥0.5; 30 genes which were regulated by these regions were annotated, 3 of which were found copy number deletion and hypomethylation simultaneously, including Ebp,Otud5,Maoa.4. Compared with normal embryos, neuroepithelial cell proliferation was decreased (P<0.05), and cellular apoptosis was increased (P<0.05) in NTD embryos; Compared with normal brains, expression of caspase3 protein was increased (P<0.05).Conclusions1. In the study, we established NTD animal model by anti-folate MTX inhibiting folate metabolism pathway, which concluded that folate dysmetabolism, not just folate deficiency, was vital for development of NTDs. 2. Folate dysmetabolism induced by MTX led to genomic DNA copy number variation and methylation alterations, resulting in genomic instability, and chromosome X was most susceptible. Genomic instability might be molecular mechanism of NTDs.3. NTDs neuroepithelial cell proliferation decreased, probably resulting from reduced DNA/RNA synthesis, and neuroepithelial cell apoptosis increased, probably resulting from genomic instability. Imbalance of neuroepithelial cell proliferation and apoptosis might lead to NTD eventually.