The Study Of MiR-130a-5p Regulating Foxa2 In Pathogenesis Of Congenital Diaphragmatic Hernia Associated Pulmonary Hypoplasia

Background: Congenital diaphragmatic hernia(CDH)is a rare congenital developmental abnormality,which causes a series of pathophysiological changes of respiratory system caused by abdominal organ herniation into the thorax,about 1/3300 of incidence in live births.With the development of prenatal diagnosis,intrauterine intervention,neonatal intensive care and surgical technology,the survival rate of children with diaphragmatic hernia has improved significantly.However,CDH-associated pulmonary hypoplasia and persistent pulmonary hypertension remain the main factors for poor prognosis in children with severe diaphragmatic hernia.Meanwhile,epidemiological studies suggest that simply repairing diaphragmatic defects and removing the compression of abdominal organs cannot effectively improve the prognosis of children with severe diaphragmatic hernia.Therefore,it is particularly important to study the molecular mechanism of CDH-associated pulmonary hypoplasia,which can provide a new means of diagnosis and treatment,and effectively improve the prognosis of children with CDH.Embryonic lung development is a complex coordinated process requiring cell differentiation and interaction between respiratory epithelial cells and the surrounding mesenchymal environment.CDH-associated pulmonary hypoplasia is characterized by fewer effective pulmonary surface areas,fewer distal branches of pulmonary parenchyma and fewer alveoli.The alveolar wall is thickened,and the interstitial components of the lung are abnormal,while compliance is decreased.Morphological characteristics of branching arrest or delay in the pseudoglandular stage of fetal lung development are observed in patients with severe fatal CDH.Several evolutionarily conserved signaling pathways are involved in different stages of embryonic lung development.In particular,fibroblast growth factor,bone morphogenetic protein,wingless secretory glycoprotein(WNT)and Hedgehog/Gli signaling are involved in lung morphogenesis and epithelial differentiation.In addition,a good balance and interaction between these signaling pathways and key transcription factors of lung development,including TTF1,GATA6 and Foxa2,are essential for normal lung morphogenesis.The Foxa transcription factor family has a wide range of functions in embryonic development,cell cycle regulation,immune regulation and so on.Foxa2 expression in the lungs is limited to a subset of respiratory epithelial cells.Foxa2 is a key transcriptional regulator in airway epithelial cell differentiation and lung development,and it contributes to normal branching and cell commitment.Transcription factors and signaling events mediating early lung formation have been extensively studied.Foxa2 regulates Shh and Shh-dependent genes in respiratory epithelial cells,thereby affecting the expression of genes required for lung branching.We compared the lung tissues of fetal rats with and without CDH by proteomic analysis and found that Foxa2 expression was downregulated in CDH lung tissue.However,the temporal and spatial expression of Foxa2 in CDH lung tissue and its regulatory mechanism in lung development are still unclear.Although about 10% of CDH patients have chromosomal abnormalities,the genetic causes of 85% of CDH patients are still unclear.The inconsistency of monozygotic twins(one fetal diaphragmatic hernia)also emphasizes the importance of considering epigenetic factors.MicroRNAs(miRNAs)are important epigenetic regulators,which are essential for embryogenesis and organogenesis.Analysis of the expression of mi RNAs at different developmental stages of human and rat lung morphogenesis has shown that the function of miRNAs is evolutionarily conserved to some extent.Dicer is a key component in the processing of miRNAs,and its inactivation during lung development has been found to lead to inhibition of lung epithelial branching.Therefore,miRNAs regulating the activity of Foxa2 are considered major molecular mediators in lung development.Objective:By constructing nitrofen-induced CDH rat model,based on proteomic data and bioinformatics analysis,to investigate the role of mi R-130a-5p/Foxa2 axis in nitrofen-induced CDH-associated pulmonary hypoplasia through ex vivo lung explants and to evaluate its potential role in prenatal treatment in vivo.Method:1.To construct the nitrofen-induced CDH rat model: female SD pregnant rats were randomly divided into CDH group(CDH)and control group(CON).On embryonic 8.5 day(E8.5),the pregnant rats in CDH group were given 100 mg of nitrofen(dissolved in 1 ml olive oil)by gavage,while the pregnant rats in CON group were given 1 ml of olive oil only.The fetal lung tissues of rats were collected on E13.5,E15.5,E17.5,E19.5 and E21.5 days.Fetal rat lung tissues from CDH group and CON group on day E17.5 were selected for TMT proteomics sequencing,and cluster analysis(GO and KEGG)were performed on differentially expressed proteins.2.The temporal and spatial expression of Foxa2 was detected by Real-time PCR,western blot and immunohistochemistry.The expression difference and localization of Foxa2 and lung epithelial cell markers in CON group and CDH group were detected by immune-fluorescence.The changes of cell proliferation and apoptosis were detected by immune-histochemistry and Western blot.3.Interaction between mi R-130a-5p and Foxa2 and its expression in fetal rat lung: Bioinformatics softwares were used to predict mir-130a-5p binding to Foxa2 mRNA and to identify the binding sites.We constructed Foxa2 wild-type and mutant luciferase vectors,which were co-transfected with mir-130a-5p mimics in 293 T cells,and detected the fluorescence value by luciferase experiment.Real-time PCR and in situ hybridization were used to detect the spatiotemporal expression of mir-130a-5p during embryonic lung development.4.The development of lung was evaluated by calculating the number of terminal branches and the surface area of lung tissue after adding Foxa2 or / and mir-130a-5p in the culture environment.Meanwhile,EdU and Western blot were used to detect the proliferation and apoptosis of lung tissue.Real-time PCR and western blot were used to detect the downstream Shh/Gli1 signaling pathway in cultured lung tissues.5.To construct the lung epithelial cell lines with Foxa2 overexpression and knockdown,to detect the proliferation ability of the lung epithelial cells with Foxa2 overexpression and knockdown by CCK-8 and EdU assay,and to detect the apoptosis ability of the lung epithelial cells with Foxa2 overexpression and knockdown by flow cytometry.To construct a lung epithelial cell line with mir-130a-5p overexpression and knockdown,CCK-8 and edu experiments were used to detect the proliferation ability of lung epithelial cells with mir-130a-5p overexpression and knockdown,and flow cytometry was used to detect the apoptosis ability of lung epithelial cells with mir-130a-5p overexpression and knockdown.The downstream Shh/Gli1 signaling pathway was detected by Real-time PCR and Western blot after over expression of mir-130a-5p and Foxa2 in lung epithelial cells.Result:1.TMT proteomics sequencing of fetal rat lung tissue.A total of 79 differentially expressed proteins(DEPs),of which 50 were up-regulated and 29 were down regulated in CDH group.GO enrichment analysis revealed that DEPs were enriched in important biological processes such as protein activation cascade,complement activation,and regulation of epithelial-to-mesenchymal transition.KEGG enrichment analysis showed that the enriched pathways include apoptosis,phospholipase D signaling pathway,HIF-1 signaling pathway,complement and coagulation cascade,Glycerolipid metabolism,and so on.Among them,the expression of Foxa2 protein was down-regulated in the fetal lung tissue in CDH group.Through online software STRING analysis,it was found that there is a mutual regulatory mechanism between Foxa2,Shh and Gli1 molecules.Therefore,we chose Foxa2 and Shh signaling pathways as research objects to further explore their expression changes in nitrofen-induced CDH rat model and their effects on lung development.2.Temporal and spatial expression of Foxa2 in embryonic lung development.In the CDH rat model,on E15.5,the expression of Foxa2 at the mRNA level was not significantly different from that of the control group,but at the protein level there was a significant difference;on E17.5,E19.5,and E21.5,The expression of Foxa2 in CDH group was significantly lower than that in control group.Immunohistochemical results suggest that Foxa2 expression is limited to airway epithelial cells(proximal bronchial epithelial cells)in the early stages of embryonic lung development(E13.5 and E15.5)and is lower in CDH group than in control group.In the later stage,during the tubule phase(E19.5)and vesicle phase(E21.5),Foxa2 expression was significantly reduced in the distal bronchial epithelial cells of CDH group.The detection of lung tissue at the vesicle stage(E21.5)indicated that the proliferation of lung epithelial cells in CDH group had decreased and apoptosis increased.3.Interaction between miR-130a-5p and Foxa2 and its expression in fetal rat lung: Bioinformatics softwares were used to predict the binding site of Foxa2 to miR-130a-5p and verified by luciferase experiments.In the lung tissue of CDH rat model,miR-130a-5p was significantly overexpressed in the lung tissue of CDH group,and the expression was opposite to that of Foxa2.In situ hybridization results suggest that miR-130a-5p expression in the epithelial lining of the proximal airway is higher than that in the distal terminal vesicles during the tubule(E19.5)and vesicle(E21.5)stages of lung development cell.4.The effect of miR-130a-5p/Foxa2 axis on lung morphogenesis in vitro.In the in vitro culture environment of embryonic lung tissue,after increasing the expression of Foxa2,the morphogenesis of lung tissues in CDH group was partially improved,including increased number of peripheral airway buds and explant surface,enhanced epithelial cell proliferation,and weaker apoptosis.Increased expression of miR-130a-5p can partially attenuate the lung development improved by Foxa2.At the same time,the Shh signaling pathway activation was evaluated by detecting Shh and Gli protein levels.In the CDH group,the expression levels of Shh and Gli1 increased after overexpression of Foxa2,and the expression levels of Foxa2 after overexpression of miR-130a-5p Decreased,while the activation of the Shh signaling pathway was partially inhibited.5.The effect of miR-130a-5p/Foxa2 axis on the proliferation and differentiation of lung epithelial cells by regulating the Shh/Gli1 signaling pathway.In human lung epithelial cell line(BEAS-2B),overexpression of mi R-130a-5p significantly inhibited the expression of Foxa2.The results of CCK-8 and EdU experiments showed that the cell proliferation ability increased after overexpression of Foxa2,and the flow cytometry experiment showed that the apoptosis rate decreased after overexpression of Foxa2.CCK-8 and EdU experiments showed that miR-130a-5p knockdown enhanced cell proliferation and flow cytometry showed that miR-130a-5p knockdown reduced apoptosis.Recovery experiments confirmed that miR-130a-5p regulates the proliferation and apoptosis of lung epithelial cells by inhibiting the expression of Foxa2.Conclusion: 1.Based on the proteomics results of the fetal lung tissue of CDH rats,during the lung development of CDH rat model,Foxa2 was downregulate expressed in CDH fetal lung tissue,and its expression was localized in lung epithelial cells and its subpopulations.2.miR-130a-5p was upregulate expressed in CDH lung tissue,and has the opposite trend to that of Foxa2 in CDH lung tissue,and Foxa2 targets miR-130a-5p.3.The miR-130a-5p /Foxa2 axis participates in the lung morphogenesis of CDH fetuses by regulating the Shh/Gli1 signaling pathway.4.miR-130a-5p affects the proliferation and apoptosis of lung epithelial cells by regulating Foxa2.