LncRNA H19 Regulates EZH2 To Promotes The Transformation Of Lung Epithelial Cells Into Mesenchymal Cells In Newborn Rats With BPD

Objective: Bronchopulmonary dysplasis(BPD)is one of the common and refractory diseases in premature infants.The main features are alveolar dysplasia and microvascular dysplasia,resulting in decreased pulmonary function.At present,a large number of studies have found that lung epithelial cells are the key target cells of BPD lung injury.Previous studies in our group confirmed that the expression of enhancer of zeste homolog 2(EZH2)was increased in lung tissue and the alveolar type II epithelial cells(AT-II cell)of neonatal rats with hyperoxia-induced BPD.It promoted epithelial-mesenchymal transition(EMT)and affected lung development in BPD by regulating the dynamic balance of EMT,however,the exact mechanism is unclear.Long non-coding RNA H19(LncRNA H19)is a gene related to embryonic development and has been confirmed in many related fields,including some regulatory roles in lung tissue development.Many studies have shown that H19 plays a biological regulatory role in cell proliferation and differentiation.The purpose of this study is to investigate the expression of LncRNA H19 in the lung tissues of BPD newborn rats and the regulatory effect on EZH2 and Naked Cutide Drosophila 1(NKD1),which elucidates the pathogenesis of EMT in pulmonary epithelial cells of BPD.Methods: Newborn SD rats were randomly divided into the model group(with 80% inhaled oxygen concentration,n=50)and the control group(with 21% inhaled oxygen concentration,n=50).Lung tissues were collected from the two groups at 1 day,3 days,7 days,14 days and 21 days after birth respectively,and the pathological changes of lung tissues were observed by hematoxylin-eosin(HE)staining.At the same time using the transmission electron microscope(TEM)to observe the ultrastructural changes of AEC II.The localization of EZH2 and NKD1 in AECII was determined by immunohistochemistry and immunofluorescence double staining.The levels of expression of mRNA and protein of H19,EZH2 and NKD1 in lung tissues were detected by fluorescence real-time quantitative PCR and Western blot.Using RNA-binding protein immunoprecipitation(RIP)to verify whether the binding site between H19 and EZH2 has a regulatory relationship.Results: HE staining showed that alveolar dysplasia was observed in the model group from 3 days compared with the control group.TEM showed that actin microfilaments and vacuolated lamellar bodies appeared in the AECII of the model group.Immunohistochemistry showed that EZH2 protein was clearly localized in the nucleus and cytoplasm of alveolar epithelial cells.And the expression of the model group was stronger than that of the control group on the first day(P<0.05),and reached the peak at 14 days(P<0.001).Immunofluorescence double staining showed that EZH2 and NKD1 were co-expressed with p180 in AT-II respectively.The results of real-time quantitative PCR showed that the mRNA expression level of H19 in BPD model group increased from 7 days to the control group(P<0.05),the expression level of EZH2 mRNA was higher than that in the control group(P<0.05),the expression level of NKD1 mRNA was lower than the control group from 3 days(P<0.05),and the difference of gene level between the two groups was more obvious with the extension of high oxygen exposure time.Western blot showed that compared with the control group,the expression of EZH2 protein in the model group increased from 1 day to 21 days(P<0.05).However,the expression of NKD1 protein in the model group was decreased from the first day(P<0.05),and the trend was maintained to 21 days(P<0.01).RIP confirmed the interaction between H19 and EZH2.The correlation analysis indicated a positive correlation between the expression of protein of H19 and EZH2(P<0.05),and a negative correlation between the expression of protein of EZH2 and NKD1(P<0.05).Conclusions: During the development and progression of BPD in neonatal rats,the expression of genes and proteins of H19 and EZH2 in lung tissue was up-regulated,and the peak of expression of H19 preceded EZH2.However,the expression of genes and proteins of NKD1 was down-regulated,suggesting that H19 regulates the expression by interacting with EZH2,resulting in inhibition of expression of NKD1.It also suggests that H19 mediates EMT in AECII,which causes pulmonary dysplasia and ultimately obstructing lung development.