Methylation Regulation On Hsa-let-7g In Neural Tube Defects And Changes Of Imprinting Genes In Major Developmental Diseases

Background Folic acid deficiency during pregnancy is believed to be a high-risk factor for neural tube defects(NTDs).It is believed that disturbed mi RNA regulation has been linked to the pathogenesis of NTDs in those with folate deficiency.However,the mechanism by which folic acid-regulated mi RNA influences this pathogenesis remains unclear.Our precious results have found that several mi RNAs tested reduced their methylation modifications in NTD cases,while the methylation of hsa-let-7g changed most.This provides direct evidence of the roles of interactions between DNA methylation and mi RNA level in these defects.Objectives To study the regulation mechanism of folate deficiency disturbs hsa-let-7g level in NTDs.Methods This is a case-control study.We compared the folate acid level and methylation level of hsa-let-7g,as well as their correlation,in the brain tussue between NTDs and controls.We explored whether the hsa-let-7g expression level was affected by promoter methylation;this potential association was assayed in HCT15 cells treated with 5-Aza.Specifically,we performed cell migration and proliferation analysis,which could reveal whether the overexpression of hsa-let-7g could disturb the invasion and proliferation compared with that of vector-transfected SK-N-SH cells.We also detected the expression level of hsa-let-7g potential target genes to analysis the role of hsa-let-7g in the NTDs pathogenesis.Results 1?The methylation level of hsa-let-7g promoter region in the NTD samples was significantly lower than that in the control samples.2?There was a significant positive correlation between hsa-let-7g methylation level and folic acid concentration in brain tissues of NTDs.3?In HCT-15 cell models,we confirmed that hsa-let-7g methylation directly regulates its own expression.4?hsa-let-7g,along with its target genes SMOX,may disturb the migration and proliferation of cells.Conclusions Modification of hsa-let-7g methylation,which is regulated by folate acid,could increase the hsa-let-7g expression level,and then probably involved in the NTDs by disturbed the cell migration and proliferation.Background As the major developmental diseases,the morbidity of Neural Tube Defects(NTDs),Embryonic Development Arrest and Congenital Heart Disease(CHD)are higher.They are all caused by the interplay of multiple genes as well as gene-environment interactions.Recently epigenetic modifications have become a new direction of exploring the pathogenesis of major developmental diseases.DNA methylation,one of the main epigenetic modifications,is also an important means of regulating genomic function.The imprinting gene regulates the growth and development of the embryo by labeling the biology process of parental information.Methylation differential region(DMR)in the imprinted genes plays an important role in the establishment of gene imprints.DMR deletion or abnormal methylation level of Cp G site in DMR can cause disordered expression of the entire imprinting genes cluster.The abnormal expression of imprinted genes will affect the normal genomic imprinting,and damage the genetic balance between the parents to affect the normal development of the embryo.Objectives To analysis the association between imprinting changes of imprinting genes and the major developmental diseases,in order to further explore the possible mechanisms.Methods By refering to the imprinting gene analysis resources,18 DMR sequence of the imprinting genes: GRB10?INPP5F?PEG10?MCST?NNAT?NAP1L5?NESPAS?GNAS?PLAGL1?MEST?KVDMR?SNRPN?ZIM2?NESP?H19?IGF2?IG and MEG3 were selected to study.This is a case-control study.We extracted the DNA of samples from NTDs,embryonic development arrests,CHDs and controls,and do the bisulfite modification of DNA.The imprinting genes DMRs were amplified by PCR and digested with PCR products.The DMR methylation level was analyzed by Mass ARRAY?Epi TYPER?,a high-throughput methylation assay.Results 1 ? There are significantly changes of some imprinting genes in the major developmental diseases of NTDs,embryonic development arrest and CHD.2?The mean methylation level of PEG10 and IG DMR in the NTD samples were significantly higher than that in the control samples.3?The mean methylation level of INPP5F?NESPAS and MEST DMR in the embryonic development arrest samples were significantly higher than that in the control samples.The high methylation levels of NESPAS DMR increased the risk of embryonic development arrest compared with low methylation levels.4?The mean methylation level of GRB10 and MEST DMR in the CHD samples were significantly higher than that in the control samples.The mean methylation level of INPP5F?PEG10?NAP1L5?PLAGL1?NESP and MEG3 DMR in the CHD samples were significantly lower than that in the control samples.The high methylation levels of GRB10 DMR,and low methylation levels of INPP5F?PEG10?PLAGL1?NESP?MEG3 DMR increased the risk of CHD.Conclusions 1?The methylation modifications of PEG10?IG DMR changed in the NTDs.2?The methylation modifications of INPP5F?NESPAS and MEST DMR changed in the embryonic development arrests.3?The methylation modifications of GRB10?MEST?INPP5F?PEG10?NAP1L5?PLAGL1?NESP and MEG3 DMR changed in the CHD.