The Abnormal Expresion Of RNA Metabolic Protein Of Neural Tube Defects

Objective: To study the expression profiles of proteomes in fetal brain tissue of human neural tube defects(NTDs)under high homocysteine(Hcy)levels in vivo and screen for differential expressed protein.Further,the changes of the expression levels of the differential proteins were verified in the HTL-induced chicken embryo model and in vitro HTL-treated mouse neural stem cell(NE4C)models.Explore the changes in the expression of differential proteins in high-Hcy-related NTDs samples provided a possible experimental basis for clinical prevention and reduction of birth NTDs fetals.Methods: On the one hand,we used high-level hcy brain tissue samples from existing human NTDs libraries and used the mass spectrometry-based isotopic tagging relative and absolute quantitative(iTRAQ)technology to screen differential expressed proteins in NTDs embryonic brain tissue.Through bioinformatics analysis,RNA-binding proteins associated with the occurrence of NTDs were further screened,and targeted proteins were absolutely quantified using mass spectrometry parallel reaction monitoring(PRM)technology to verify their expression.On the other hand,the homocysteine-induced chicken NTDs model was constructed.The homocysteine thiolactone(HTL)was injected into the nerve groove of chicken embryos in E2.Chicken embryos in the normal and HTL-treated groups were collected in E5.We use the PRM technology to verify the difference of RNA-associated proteins between the two groups.At the same time,RT-PCR was used to verify the mRNA levels and immunohistochemistry was used to verify the expression changes of the differential expressed proteins.In addition,the neural stem cells of NE4 C were treated with 5mM HTL at the cell level in vitro.The mRNA levels of the differential expressed proteins were verified by RT-PCR.WB and immunofluorescence were used to verify the expression of differential expressed proteins.Results:(1)A total of 6076 proteins were detected in human embryonic brain tissue.By comparing NTDs brain tissue samples with a high level of Hcy and normal control samples,a total of 448 differential expressed proteins were found with a threshold of 1.3.The number of protein increased was 214 and decreased was 234.Bioinformatics analysis showed that the differentially expressed proteins were associated with biological processes such as RNA splicing,ribosomal synthesis,RNA transport,RNA degradation and mRNA regulation.Besides,the differentially expressed proteins presented a downward trend.(2)The PRM technique was used to further verify the changes of differential expressed proteins in 4 normal and 4 NTD samples.The levels of six proteins(ALYREF,RBMX,HNRNPA1,HNRNPA3,HNRNPAB,EEF1A1)were significantly reduced in NTD samples.This is consistent with the iTRAQ results.(3)The chicken NTDs model was firstly constructed by HTL nerve groove injection.(4)In the HTL-induced chicken NTDs model,PRM analysis revealed that the spliceosome-associated RNA-binding proteins HNRNPA1 and HNRNPAB showed a decreasing trend,and mRNA expression levels also decreased.Immunohistochemistry further confirmed the low expression of HNRNPA1 and HNRNPAB.(5)In HTL-treated NE4 C cells,westernblott suggested that the expression levels of HNRNPA1 and HNRNPAB were decreased and the mRNA expression was also decreased.Immunofluorescence suggested abnormal subcellular localization of HNRNPA1 and HNRNPAB.(6)WB showed decreased expression of HNRNPA1 and HNRNPAB in NTD fetal brains.Conclusion:(1)These research suggests that high Hcy may play a new role in the pathogenesis of neural tube defects by affecting the low expression of RNA splicing pathway-related proteins HNRNPA1 and HNRNPAB,which may be a new mechanism for the occurrence of NTDs(2)The new method of 0.5mM HTL inject into neural groove successfully constructed a neural tube defect animal model of chicken embryos.