The Role And Mechanism Of DAB2IP In Congenital Diaphragmatic Hernia Associated Pulmonary Dysplasia

Background: Congenital diaphragmatic hernia(CDH)is a kind of disease due to diaphragmatic defect,resulting in abdominal contents protruding into the chest,interfering with the normal development of the lung.The incidence of CDH in neonates ranged from 0.8 to 5 / 10000 and varied with the population.In recent decades,due to the development and standardization of prenatal diagnosis,pregnancy monitoring and surgical treatment technology,the survival rate of children with CDH has an increasing trend.However,a variety of factors,such as pulmonary hypoplasia,persistent pulmonary hypertension in the newborn(PPHN)and so on,can affect the prognosis and survival of children with CDH.Therefore,it is urgent for us to find the pathogenesis of CDH associated pulmonary hypoplasia and provide theoretical support for improving the prognosis of children with CDH.In our previous study,we used nitrophenol-induced rat CDH model to analyze the proteomics of fetal lung tissue,and found that the expression of DAB2 IP was upregulated in fetal lung tissue with diaphragmatic hernia.DAB2IP(disabled homolog 2-interacting protein)is a ras GTPase activating protein(GAP),which is involved in the regulation of a wide range of signaling pathways and special signaling pathways.It is involved in the innate immune response,inflammation and cell growth inhibition,apoptosis,cell survival,angiogenesis,cell migration and maturation,and has the effect of inhibiting tumor.The expression of DAB2 IP was time-specific during the development of fetal organs.The expression of DAB2 IP in fetal lung was very low,mainly in bronchioles,located in the cytoplasm of terminal bronchioles epithelial cells.RAS(rat sarcoma)is a GTPase,which acts as a molecular switch to regulate cell proliferation,survival,growth,migration,differentiation or cytoskeleton activity.Ras protein transmits signals from extracellular growth factors by cycling between the non activated GDP binding state and the activated GTP binding state.The exchange of GTP with GDP on RAS is regulated by guanine nucleotide exchange factors(GEFs)and GTPase activating proteins(GAPs).Activated RAS(GTP-RAS)regulates a variety of cellular functions through effectors,including RAF,phosphatidylinositol 3-kinase(PI3K)and Ral guanine nucleotide dissociation stimulator(RALGDS).RAS is involved not only in tumorigenesis,but also in many developmental disorders.However,up to now,the temporal and spatial expression of DAB2 IP in diaphragmatic hernia lung tissue and its regulatory mechanism in lung dysplasia have not been studied.Objective: To investigate the role of DAB2 IP in diaphragmatic hernia related pulmonary dysplasia by using a rat model of diaphragmatic hernia induced by nitrophenol.Methods: 1.To establish a rat model of diaphragmatic hernia induced by nitrophenol.The temporal and spatial expression and localization of DAB2 IP during lung development were detected by immunohistochemistry,real-time PCR and Western blotting.2.The difference of GTP-RAS in fetal lung was detected by Pull Down test.3.The changes of RAF-MEK-ERK and PI3K-AKT signaling pathways in lung tissue were detected by Western blotting.4.The proliferation and apoptosis of lung tissue were detected by Western blotting,immunohistochemistry and immunofluorescence.5.DAB2IP overexpression plasmid and si RNA were transfected into lung epithelial cells.The difference of GTP-RAS was detected by Pull Down test.6.Western blotting was used to detect the changes of RAF-MEK-ERK and PI3K-AKT signaling pathways downstream of Ras.7.Western Blotting,CCK-8 and EDU were used to detect the proliferation and apoptosis of lung epithelial cells.Results: Compared with the control group,the transcription and expression levels of DAB2 IP were increased in the CDH group,but there was no significant change with time.The results of IHC suggested that DAB2 IP was mainly located in respiratory epithelial cells.At E19.5d and E21.5d,the number of pulmonary alveoli in the CDH group was significantly reduced and the alveolar wall was significantly thickened,suggesting that the abnormal proliferation and differentiation of respiratory epithelial cells may be related to the up regulation of DAB2 IP expression.At E17.5d and E21.5d,the content of GTP-RAS in CDH group was significantly decreased,but there was no significant difference in total-Ras.The protein phosphorylation levels of RAS-MAPK(RAF-MEKERK)and PI3K-AKT pathway were decreased in CDH group.At the same time,the expression of PCNA was decreased and the ratio of Bax / Bcl-2 was increased.The results of immunohistochemistry and immunofluorescence showed that the decrease of cell proliferation and the increase of apoptosis in the lung tissue of CDH group were mainly manifested in the lung epithelial cells.After overexpression of DAB2 IP in lung epithelial cells,there was no significant difference in the expression of total-RAS,but the content of GTP-RAS was significantly decreased.The phosphorylation levels of RAF-MEK-ERK pathway and PI3K-AKT pathway were decreased.The proliferation marker PCNA was decreased,and the apoptosis marker Bax/Bcl-2 was increased.The proliferation ability of lung epithelial cells was significantly decreased,and the percentage of proliferation cells was significantly decreased.After silencing DAB2 IP in lung epithelial cells,there was no significant difference in the expression of total-Ras and GTP-RAS.Although the phosphorylation levels of several indexes of RAF-MEK-ERK and PI3K-AKT pathway were showed a decreasing trend,there was no significant difference.The ratio of Bax/Bcl-2 was decreased,but the percentage of proliferating cells was slightly decreased.Conclusions: 1.In the rat model of congenital diaphragmatic hernia,the transcription and expression levels of DAB2 IP in lung tissue of CDH group were up-regulated,and the expression was mainly located in the respiratory tract epithelial cells.2.In the lung tissue of fetal with CDH,the level of GTP-RAS,the phosphorylation of RAS-MAPK and PI3K-AKT pathways were all decreased,and the level of proliferation decreased,and the level of apoptosis increased.3.Overexpression of DAB2 IP in human lung epithelial cells decreased the level of GTP-RAS,phosphorylation levels of RAS-MAPK and PI3K-AKT pathways,inhibited the proliferation and promoted the apoptosis of lung epithelial cells.