Mi R-26a Targeting EPHA2 Regulates MEK Pathway And SRF To Participate In The Pathogenesis Of Neural Tube Defects

Neural tube defect(NTD)is a common congenital malformation of the central nervous system,which is caused by incomplete closure of neural tube during the development of neural embryo after conception.The involvement of excessive nerve cell apoptosis and insufficient proliferation in neural tube closure has been a hot topic in the field of neural tube defects in recent years,but the pathogenesis of NTDs is still not completely clear.Bioinformatics analysis of the human neural tube defect amniotic fluid data set revealed that EPHA2 expression is down-regulated and plays an important role in axonogenesis and positive regulation of neurogenesis.NTDs are typical multifactorial events in which epigenetic modification plays an extensive role in their pathogenesis.Mi RNAs have different expression patterns during central nervous system development.We found that mi R-26 a targets EPHA2 to regulate the proliferation and apoptosis of neural cells,preliminarily verified this conjecture,and explored its downstream mechanism.Chapter 1: Bioinformatics analysis of NTD related mechanism and clinical correlation verificationObjectives: The purpose of this chapter is to find possible interaction factors related to the pathogenesis of human neural tube defects from the chip data from GEO database(http://www.ncbi.nlm.nih.gov/geo/),analyze their relationship and conduct preliminary clinical tissue sample verification.Methods: The microarray of amniotic fluid samples with human neural tube defects was screened from GEO database,using limma package of R language(version 4.1.2),according to P< 0.05 and |Log2FC|>1 criteria for differential gene analysis.Cluster Profiler packages were used for GO and KEGG enrichment analysis of differential genes to search for differential genes with important physiological functions in the development of the nervous system.Protein-protein interaction analysis of target genes was performed using STRING,Cytoscape and Gene Mania.Targetscan,mi Walk,mi RDB and DTANm T were used to predict online the mi RNAs that might be bound by the above genes,and we found that hsa-mi R-26a-5p,hsa-mi R-26b-5p and hsa-mi R-1297 were in the largest intersection.The brain and spinal cord nerve tissues of the induced fetus(included 17 cases with NTDs diagnosed by ultrasound and 16 cases requiring abortion for non-medical reasons confirmed by autopsy)were collected,and RNA was extracted for real-time quantitative PCR verification.The proteins were extracted and western blot was performed to detect the expression of the above factors in clinical tissue samples.Results: We screened DSG1,RPS4Y1,KRT24,MMP7,EPHA2,PTPN3 and other down-regulated genes with significant differential expression from GEO chip GSE4182 and GSE101141 data sets.GO analysis of differential genes revealed that they play an important role in axon pathfinding and play a positive regulatory role in neurogenesis.KEGG enrichment analysis showed that ERK/MAPK(MEK)pathway was the most important signal transduction pathway.Transcription factor SRF is located downstream and plays a role in cell proliferation and differentiation.PPI analysis showed that EPHA2 is important in differentially expressed genes in NTDs’ amniotic fluid.Protein-protein interaction analysis revealed an association between EPHA2,ERK1/2 and SRF.Mi RNAs that have a target relationship with EPHA2 were predicted online by Targetscan,mi Walk,mi RDB and DIANAm T and the intersection was taken.Mi R-26 a closely related to nervous system development was selected from the mi RNAs with the most intersection through literature mining.Literatures suggest that mi R-26 a may be involved in nerve growth and development.The results of clinical tissue experiments showed that the levels of mir NA-26 A were significantly increased and the levels of EPHA2 and ERK1 were significantly decreased in the neural tube defect group compared with the normal control group.Correlation analysis showed that mi RNA-26 a was negatively correlated with EPHA2 and ERK1 m RNA levels,while EPHA2 and ERK1 m RNA levels were significantly positively correlated.Conclusion: Bioassay data suggest that mi R-26 a targeting EPHA2 regulates SRF via MAPK pathway and may play a role in nerve cell proliferation and apoptosis.Clinical tissue experiments confirmed that the expression of mi R-26 a,EPHA2 and ERK1 was related to the pathogenesis of neural tube defects.The expression of mi R-26 a was up-regulated and the expression of EPHA2 and ERK1 was down-regulated in NTDs fetal neural tissues.Chapter 2: Mi R-26 a and MEK pathway regulates the proliferation and apoptosis of neural cellsObjectives: The biological functions of mi R-26 a and MEK pathway in the growth and development of mammalian neural cells were investigated on mouse neural stem cell NE-4C cells and human astrocytoma U87 MG cells.Methods: Mi RNAs and si RNAs were transfected into NE-4C cells and U87 MG cells using Lipo3000 plasmid vector,respectively,to overexpress or inhibit mi R-26 a expression in these two cells.ERK/MAPK(MEK)inhibitor PD98059 was added to inhibit ERK phosphorylation in MAPK pathway.The cells were divided into six groups: NC,mimic NC,inhibitor NC,mi R-26 a mimic,mi R-26 a inhibitor and PD98059,respectively.CCK8 assay was used to detect the proliferation ability of the two cell lines.Annexin V/7-AAD labeled cells were detected by flow cytometry and the apoptosis rate of each group was calculated.QRT-PCR and WB were used to detect the level of caspase9 and caspase3 in each group.Results: In NE-4C and U87 MG cells,overexpression of mi RNA-26 a decreased cell proliferation(P<0.05),increased the rate of early apoptosis(P<0.01),and increased caspase9/3 levels(P<0.01 or P>0.05);Inhibition of ERK phosphorylation significantly reduced cell proliferation activity,increased the rate of early apoptosis and caspase9/3 levels(P<0.01 or P>0.05);Inhibition of mi RNA-26 a enhanced cell proliferation activity(P<0.05),decreased the rate of early apoptosis and caspase9/3 levels(P<0.05 or P>0.05).Conclusions: Mi R-26 a and ERK/MAPK(MEK)pathway are both involved in regulating the proliferation and apoptosis of neural cells.Chapter 3: Mi R-26 a targeting EPHA2 regulates MEK pathway and SRFObjectives: To preliminarily explore the mechanism of mi RNA-26 a targeting EPHA2 to regulate MEK pathway and SRF,which may be related to the pathogenesis of neural tube defects.Methods: Target Scan Human7.1 was used to predict the binding site of mi R-26 a and EPHA2,and EPHA2 wild-type and mutant plasmid vectors were constructed and verified by sequencing.Double luciferase reporter gene detection analysis was performed to verify the binding site.Mi RNAs and si RNAs were transfected into NE-4C cells and U87 MG cells using Lipo3000 plasmid vector,respectively,to overexpress or inhibit mi R-26 a expression in these two cells.ERK/MAPK(MEK)inhibitor PD98059 was added to inhibit ERK phosphorylation in MAPK pathway.The cells were divided into six groups: NC,mimic NC,inhibitor NC,mi R-26 a mimic,mi R-26 a inhibitor and PD98059,respectively.The levels of mi RNA-26 a and EPHA2,ERK1,SPF gene were detected by QRT-PCR and WB.Results: After transfection of mi RNA-26 a,reported fluorescence of hsa-EPHA2-WT and mus-EPHA2-WT was significantly down-regulated(P <0.01),while there was no statistical difference in reported fluorescence in hsa-EPHA2-MUT and mus-EPHA2-MUT compared with the negative control group(P>0.05).In NE-4C cells,compared with the mimics NC group,m RNA and protein levels of EPHA2 and SPF and phosphorylation of ERK1 in the mi RNA-26 a mimics group were decreased(P<0.01);compared with the inhibitor NC group,m RNA and protein levels of EPHA2 and SPF and phosphorylation of ERK1 in the mi RNA-26 a mimics group were increased(P<0.05 or P>0.05);compared with the NC group,the phosphorylation level of ERK1 and the m RNA and protein levels of SRF in PD98059 group were decreased(P<0.01).In U87 MG cells,compared with the mimics NC group,m RNA and protein levels of EPHA2 and SRF and phosphorylation of ERK1 in the mi RNA-26 a mimics group were decreased(P<0.01);compared with the inhibitor NC group,the m RNA and protein levels of EPHA2 showed an increasing trend(P>0.05),the phosphorylation level of ERK1 and the m RNA and protein levels of SPF were increased(P<0.01)in mi RNA-26 a inhibitor group;compared with the NC group,the phosphorylation level of ERK1 and the m RNA and protein levels of SPF in PD98059 group were significantly decreased(P<0.01).Conclusions: Mi RNA-26 a targets EPHA2 to regulate ERK1 phosphorylation and SRF,which may be involved in the pathogenesis of NTDs.