| Objective:Neural tube defects（NTDs）are severe congenital defects caused by incomplete neural tube closure.The harm of NTDs is serious and its causes are complicated.Micro RNA（miRNA）plays an important role in neural tube development,and its role in the occurrence of NTDs remains unclear.It is of great significance to further study the pathogenesis of NTDs from the epigenetic perspective of miRNA.The purposes of this study:1 From the miRNAomics level,systematically explore the temporal changes of miRNA expression profiles in the embryonic neural tubes of retinoic acid（RA）-induced NTDs,and screen for the important differentially expressed miRNAs related to NTDs.2 To study the expression of miR-10a-5p in NTDs and normal mice embryonic neural tubes,using neural stem cells（NSCs）as a cell model to explore its function and mechanism in the occurrence of RA-induced NTDs.Methods:1 Construct the NTDs mice models and small RNA sequencing（s RNA-seq）.1.1 C57BL/6 mice were treated as experimental animals.At embryonic day 7.5（E7.5）,mice in NTDs model group were gavaged with 28 mg/kg（body weight）of RA and the mice in control group were gavaged with the same dose of sesame oil.1.2 On E8.5,E9.5 and E10.5（the stage of neural tube closure）,tissues of mice embryonic brain vesicle were collected.Illumina Hi Seq TM2000 was used for s RNA sequencing.Mi RNA expression libraries of the normal group（Brc8.5,Brc9.5 and Brc10.5）and the NTDs group（Bra8.5,Bra9.5 and Bra10.5）were obtained.2 Analyze the miRNA expression profiles in NTDs mice embryos.2.1 Fold changes of normalized expression of miRNAs（log2 Ratio Bra/Brc）and P values in control group and NTDs group libraries were calculated.Mi RNAs with|log2Ratio|≥1 and false discovery rate（FDR）≤0.001 were defined as significantly differentially expressed miRNAs（DEmiRNAs）in the two groups.Mi RNAs that were differentially expressed in library pairs of Bra8.5-vs-Brc8.5,Bra9.5-vs-Brc9.5 and Bra10.5-vs-Brc10.5 were obtained,and the common differentially expressed miRNAs（co-DEmiRNAs）were further obtained through intersection analysis.2.2 Targetscan was used to predict the target genes of miRNA.GO and KEGG pathway enrichment analyses were conducted to understand the biological processes and signal metabolic pathways involved in the candidate target genes.2.3 Some screened NTDs-related differentially expressed miRNAs were verified by RT-q PCR.3 Mi R-10a-5p,the upregulated differentially expressed miRNA with the highest expression abundance and maximum difference fold change in NTDs mouse embryos based on the s RNA-seq date,was selected as the target of this part of study to further investigate its effect on the biological function of nerve cells.3.1 RT-q PCR was used to detect the spatial and temporal expression pattern of miR-10a-5p in mice embryos of NTDs.3.2 NSCs and N2a were transfected with miR-10a-5p overexpressed lentivirus（LV-miR-10a）and miR-10a-5p mimics（mim-10a）respectively,and the expression level of miR-10a-5p was detected by RT-q PCR.3.3 Brd U and CCK-8 assay were used to detect the effect of miR-10a-5p overexpression on cell proliferation.The effects of miR-10a-5p overexpression on cell cycle and apoptosis were detected by flow cytometry（FCM）.The effect of miR-10a-5p overexpression on the differentiation of NSCs into neurons and astrocytes was detected by cellular immunofluorescence,and MAP2 and GFAP positive cells were counted respectively.4 Study on the mechanism of miR-10a-5p regulating CHL1 in NTDs.4.1 Bioinformatics analysis was used to predict the target genes of miR-10a-5p,and Chl1 with high expression and important function in the nervous system was screened as the candidate target gene.4.2 Double-luciferase report assay was used for verification of target genes.4.3 The protein expression level of CHL1 was detected by western blot（WB）in NTDs mice embryonic tissues induced by RA.The expression of CHL1 in embryonic brain slices was detected by immunofluorescence.4.4 After transfection with LV-miR-10a,RT-q PCR and WB were used to detect the m RNA and protein expression level of target genes Chl1 in NSCs.4.5 After transfection with CHL1 si RNA,RT-q PCR and WB were used to detect the m RNA and protein expression level of CHL1 in NSCs.Brd U assay was used to detect the effect of CHL1 knockdown on cell proliferation.The effects of CHL1 knockdown on cell cycle and apoptosis were detected by FCM.The effect of CHL1 knockdown on the differentiation of NSCs into astrocytes was detected by cellular immunofluorescence,and GFAP positive cells were counted.4.6 After LV-miR-10a transfection into NSCs for 24 hours,the AV-CHL1 containing the CHL1 open reading frame（ORF）lacking the 3?UTR sequence was transfected into NSCs for restored the expression of CHL1.WB was used to detect the protein expression level of CHL1.Brd U assay was used to detect the effect of CHL1 reexpression on cell proliferation.The effect of CHL1 reexpression on the differentiation of NSCs into astrocytes was detected by cellular immunofluorescence,and GFAP positive cells were counted.4.7 The expression levels of MAPK pathway proteins were detected by WB in NTDs mice embryonic tissues induced by RA.The expression of Ki67 in slices was detected by immunohistochemistry.Terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling（TUNEL）was used to detect the cell apoptosis in the sections.4.8 After transfection with CHL1 si RNA in NSCs,the proteins expression levels of the downstream ERK/MAPK signaling pathway of the CHL1 were detected by WB.4.9 After LV-miR-10a transfection into NSCs for 24 hours,the honokiol was added into NSCs to restore the expression of p-ERK1/2.WB was used to detect the protein expression level of ERK1/2.Brd U assay was used to detect the effect of p-ERK1/2reexpression on cell proliferation.The effect of p-ERK1/2 reexpression on the differentiation of NSCs into astrocytes was detected by cellular immunofluorescence,and GFAP positive cells were counted.Results:1 Construct the NTDs mice models and s RNA-seq.1.1 On E8.5,the embryonic nerve folds in the NTDs group had abnormal morphology and delayed closure compared with the control group.On E9.5 and E10.5,the embryonic brain structures of mice in the NTDs group were deformed and the neural tubes were not closed.These show that this experimental method successfully constructed the RA-induced NTDs mice models,which can well simulate the clinical NTDs phenotype of anencephaly.1.2 The quality control of the extracted RNAs,c DNA libraries and sequencing datas indicated that they were of high quality and could be used for subsequent bioinformatics analysis.2 Analyze the miRNA expression profiles in NTDs mice embryos.2.1 A total of 241 miRNAs（11 miRNAs upregulated and 230 miRNAs downregulated（11/230））were identified as differentially expressed in the library Bra8.5-vs-Brc8.5comparison.The number of DEmiRNAs were found in other stages with 271（47/224）and213（46/167）in Bra9.5-vs-Brc9.5 and Bra10.5-vs-Brc10.5,respectively.2.2 Targetscan was used to predict candidate target genes with differentially expressed miRNA.Then target genes were filtered by combining s RNA-seq with m RNA-seq data of the research group,and the filtered differentially expressed miRNAs and target genes were obtained.There were 232 miRNA-m RNA pairs which contained 62 miRNAs and 147genes in Bra8.5-vs-Brc8.5;1223 miRNA-m RNA pairs which contained 115 miRNAs and 779 genes in Bra9.5-vs-Brc9.5;and 1530 miRNA-m RNA pairs which contained 84miRNAs and 853 genes in Bra10.5-vs-Brc10.5.2.3 After the GO functional enrichment analysis of the filtered target genes of miRNAs,we found that the biological processes involved in the target genes at various stages mainly include neuron fate commitment,neuron differentiation,central nervous system development,neurogenesis,nervous system development and so on.All target genes and corresponding miRNA-m RNA pairs enriched in GO term“central nervous system development”were analyzed,and 19 miRNA-m RNA negative regulation pairs were screened.2.4 KEGG pathway enrichment analysis of the filtered target genes of miRNAs showed that these genes were mainly involved in ECM-receptor interaction,pathways in cancer,neuroactive ligand-receptor interaction and so on.2.5 The intersection analysis among Bra8.5-vs-Brc8.5,Bra9.5-vs-Brc9.5 and Bra10.5-vs-Brc10.5 was performed,and 41 co-expressed DEmiRNAs were obtained.The hierarchical clustering analysis of 41 DEmiRNAs divided the miRNA subgroups into 5categories.The expression levels of 5 of 41 miRNAs at E10.5 mice embryos were verified by RT-q PCR,and their expressions levels were consistent with the sequencing results.2.6 Co-analysis was performed on 41 DEmiRNAs and 196 differentially expressed m RNAs（DEGs）.We screened 8 miRNA-m RNA pairs,and their expression patterns were negatively correlated at three time points.3 Effect of abnormal expression of miR-10a-5p on the biological function of nerve cells.3.1 RT-q PCR results showed that the expression levels of miR-10a-5p in NTDs mice embryos were significantly higher than those of the normal group.3.2 RT-q PCR results showed that the expression levels of miR-10a-5p were significantly increased after transfected with LV-miR-10a and mim-10a in NSCs and N2a respectively.3.3 Brd U and CCK-8 results showed that miR-10a-5p overexpression inhibited cell proliferation.FCM results showed that overexpression of miR-10a-5p blocked the cell cycle,and the cells were trapped in the G1 phase and could not enter the S phase.FCM results showed that overexpression of miR-10a-5p induced cell apoptosis.Immunofluorescence results showed that the ability of NSCs to differentiate into astrocytes was significantly lower than that of the control group when miR-10a-5p was overexpressed.4 Study on the mechanism of miR-10a-5p regulating CHL1 in NTDs.4.1 Chl1 was screened as candidate target gene of miR-10a-5p by bioinformatics analysis.4.2 Dual-luciferase reporter assay confirmed that Chl1 was the target gene of miR-10a-5p.4.3 WB results showed that the protein expression levels of CHL1 weas decreased in NTDs mice embryonic tissues induced by RA.Immunofluorescence results showed that the expression levels of CHL1 in NTDs embryonic brain slices were decreased.4.4 RT-q PCR and WB results showed that the m RNA and protein expression of CHL1 were decreased after miR-10a-5p overexpression in NSCs.4.5 RT-q PCR and WB results showed that the m RNA and protein expression of CHL1 were decreased after transfection with si R-CHL1 in NSCs.Brd U results showed that CHL1 knockdown inhibited cell proliferation.FCM results showed that CHL1 knockdown blocked the cell cycle,and the cells were trapped in the G1 phase and could not enter the S phase.FCM results showed that CHL1 knockdown induced cell apoptosis.Immunofluorescence results showed that the ability of NSCs to differentiate into astrocytes was significantly lower than that of the control group after CHL1 knockdown.4.6 WB results showed that the protein expression of CHL1 was restored after co-transfection with LV-miR-10a and AV-CHL1.Brd U results showed that CHL1reexpression significantly abrogated the activity of miR-10a-5p to inhibit the cell proliferation.Immunofluorescence results showed that CHL1 reexpression significantly abrogated the effect of miR-10a-5p on the differentiation of NSCs into astrocytes.4.7 WB results showed that the expression levels of MAPK pathway proteins were decreased in NTDs mice embryonic tissues induced by RA.Immunohistochemistry results showed that the expression of Ki67 in NTDs slices was decreased.TUNEL results showed that the cell apoptosis in the sections of NTDs increased compared with the control group.4.8 WB results showed that the proteins expression levels of downstream ERK/MAPK signaling pathway of the CHL1 were decreased after transfection with si R1-CHL1 in NSCs.4.9 WB results showed that the protein expression level of p-ERK1/2 was restored when the honokiol was added into NSCs after transfection with LV-miR-10a for 24 hours.Brd U results showed that p-ERK1/2 reexpression significantly abrogated the activity of miR-10a-5p to inhibit the cell proliferation.Immunofluorescence results showed that p-ERK1/2 reexpression significantly abrogated the effect of miR-10a-5p on the differentiation of NSCs into astrocytes.Conclusions:1 By means of differential expression analysis and functional enrichment analysis on the miRNA expression profile of NTDs mice embryos,we identified NTDs-related miRNAs,and the biological functions of predicted target genes were analyzed.RT-q PCR was used to verify the expression of certain miRNAs.We also identified the abnormally high expression of miR-10a-5p in RA-induced NTDs embryos,which may be closely related to the occurrence of NTDs.2 MiR-10a-5p plays a teratogenic role in RA-induced NTDs,which inhibits downstream MAPK signaling pathway by targeting Chl1,resulting in cell proliferation,apoptosis and differentiation disorder,and affecting neural tube development. |
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