Construction And Analysis Of Folic Acid Deficiency Human Neural Crest Cell Model And Mouse Model

BackgroudsNeural tube defects(NTDs)are a spectrum of birth defects that includes anencephaly,spina bifida(meningomyelocele),craniorachischisis according to the position and extent of the defects.The neural crest cells(NCCs)are a group of heterogenous multipotent cells which arises from the dorsal midline of the neural tube during embryonic development.It is transient and also can migrate extensively and differentiate into diverse derivatives under specific circumstances.Successful morphogenesis of the neural tube in the developing embryo is dependent on the intricate coordination of the processes that involves NCCs approriate differentiation,migration,proliferation,and apoptosis.Due to the ethical and technical issues,directly observation of the human embryonic development in vivo or obtaining the NCCs are quite difficult,and there are still species variances among the human and animal models,therefore,building the human-origined NCCs model in vitro is quite necessary for further studies of NTDs mechanisms and medicine screening for disease prevention and cure.Folic acid(FA)is a water-soluble B vitamin,which is chemically consist of a pteridine moiety,para-aminobenzoic acid and L-glutamic acid,therefore it is also called pteroylglutamic acid(PGA).Inside the cell,folic acid and dihydrofolic acid(DHF)enters the folate cycle after being reduced by dihydro folate reductase(DHFR)into its biologically active form,tetrahydrofolate(THF),which is capable of accepting a one-carbon unit(OCU)at N5 or N10 positions.In this way,FA take part in the one-carbon metabolism(OCM),which will affect DNA/RNA/protein/lipids methylation,histone modification and other epigenetic regulations.Epigenetic modifications are defined as mechanisms that regulate gene expression without altering the underlying sequence of DNA.Epigenetic mechanisms comprise DNA methylation,histone modifications which includes acetylation,methylation,ADP-ribosylation,ubiquitination,and phosphorylation among others,through which will determine conformational changes in the chromatin—another epigenetic mechanism called chromatin remodeling.What's more,miRNAs and RNA binding proteins(RBPs)have also been recognized as the epigenetic mechanisms for its capacity to regulate specific target mRNAs.RBPs are an extensive class of proteins that can recognize and bind to specific sequences of RNA and regulate their functions;mechanisms of posttranscriptional control regulated by RBPs including capping,pre-mRNA splicing,mRNA export,mRNA stability modulation and translation regulation.ObjectivesEpidemiologic studies have show that dietary folate deficiency is associated to developmental anomalies(e.g.neural tube defects)as well as increased risk for cancers,anemia,cardiovascular diseases,among others.Maternal folate intake has reduced the incidence of human neural tube defects by 60-70%.Deficits in FA metabolism could affect cell proliferation,cell survival,transcriptional regulation,or a host of other cellular reactions;Therefore,the answer to FA-deficiency induced-NTDs is complex as folate is central to numerous cellular reactions.The goal of this study is to further the understanding of NTDs for better prevention and treatment of these malformations.Methods and Results1.NCCs derived from H9 hESCs and verificationsMethods:Using a well-established protocol,NCCs were differentiated from H9 hESCs.We used Real-time PCR to examine the mRNA expression of NCCs' markers and then using immunofluorescent staining and flowcytometry to verify the surface markers of the NCCs at protein level.Results:A population of stellate-morphology cells migrated away from the H9 hESCs clones.Both mRNA and protein level of specific markers of NCCs such as SOX9,p75,HNK1 and AP2-a are significantly higher than the H9 hESCs as control(Ctrl).2.FA deficiency NCCs modelsMethods:Three models of folic acid-deficiency are built up:mediums without FA added(FAD group),medium with FA antagonist methotrexate(MTX)supplementation(MTX group)and using shRNA to knockout reduced folate receptor(shRFC group),respectively.ELISA is used to examine the FA level in the NCCs in each group.CCK8(cell counting kit 8)analyze the cell proliferation,Transwell chambers are used to measure the migration ability of NCCs,The MuseTM Annexin V/7-AAD assay is used to evaluate the apoptosis of NCCs.Results:It turns out that FA deficiency impairs cell proliferation and migration,and cause elevation of apoptosis,thess changes in biological traits may attributed to the reasons for NTDs occurrance.3.FA deficiency mouse modelsMethods:C57BL/6J mice are divided into FAD groups and control groups at random.The controls are subjected to AIN-93G diets while the FAD groups are treated with modified AIN-93G diets minus FA meanwhile plus 1%succinylsulfathiazole to suppress FA absorption in intestines.FAD treating lasts for 8,10,12 weeks respectively.The mice are mated in the ratio of 1:2-1:1,the day seeing the plug is recorded as E0,the embryos are harvested in dpc10.5,meanwhile the blood is obtained by eye-removing method,the FA level in mouse serum is measured by ELISA.Pictures of embryos are taken to evaluate the ratio of NTDs and resorptions.Crown-rump length(CRL)is measured by Image J.Results:ELISA shows that the serum FA level significantly decreased and NTDs embryos also appeared from 8w group.The malformation incidence is certainly higher in FAD group,however the resorption rate seems irrelavant to the FAD conditions.The CRL of FAD group is significantly lower than the Ctrl group.4.The relationship between FAD and RBPsMethods:In this chapter,we use bioinformatic tools to explore possible mechanisms of FAD-induced NTDs.FAD gene chip that already exists in GEO database is used,Gene ontology(GO)enrichment analysis and KEGG pathway analysis are done to determine the possible relevant pathways that affect the occurrence of NTDs.The significantly differently expressed genes are screened out and then compared to the human RBPs,which have 38 in common,we then verify the protein expression level of the RBPs in all three FAD models using Real-time PCR and further confirm the protein expression with western blot.Then we use Real-time PCR to evaluate the mRNA of RBPs in mouse FAD model to see whether the RBPs also increase in vivo.Finally,the RBPs that express differently both in vivo and in vitro at the same time are knocked down by shRNAs in human NCCs FAD model to further inspect the relationship of RBPs with the biological traits changes of NCCs under FAD condition.Results:38 RBPs that show increased expression are screened out,and we further verify these RBPs' mRNA expression level in our experiment models using Real-time PCR and narrow them down to 4 RBPs in FAD group(HNRNPM,HNRNPC,LARP6,TRMT1)and 2 in shRFC group(PPIL4,RCAN2)but not in MTX group.The results show that both mRNA and protein levels are significantly higher compared to the control group.There are 2 RBPs in intersection with mouse and human NCCs FAD model,namely HNRNPC,LARP6 and 1 in human NCCs-shRFC group(RCAN2).Then we investigate the effects of RBPs on cell biological traits under FAD culture by knockingdown these three RBPs respectively.Knocking down of hnRNPC under FAD culture medium can promote NCCs proliferation but inhibit migration and enhance NCCs apoptosis,while LARP6 knockdown-NCCs exhibit significantly increased proliferaton ability and decreased apoptosis but Transwell assay indicate no significant effects on NCCs migration ability compared with control group.What' worth noticing is that knocking down of RCAN2 promotes NCCs' proliferation as well as migration and significantly inhibits NCCs apoptosis under FAD conditions.Conclusions1.Three models human neural crest cell(NCC)under folic acid deficiency(FAD)conditions are built and they prove that FAD negatively affects the cell proliferation and migration abilities and promote NCCs apoptosis.2.RBPs express high both in human NCCs FAD model in vitro and in mouse FAD model in vivo,suggests that RBPs are correlated to the abberation of NCCs'proliferation,migration abilities and apoptosis under FAD conditions.