Mechanism Of TOP2A Interactions With β-catenin To Regulate Transdifferentiation Of Alveolar Epithelial Cells In A Hyperoxia-induced Neonatal Rat BPD Model

Objective:In this study,we will search the expression of DNA topoisomerase Ⅱ alpha（TOP2A）in neonatal rat bronchopulmonary dysplasia（BPD）model,explore the interaction between TOP2A and WNT/β-catenin signaling pathway,and clarify the regulatory role of TOP2A on the transdifferentiation of alveolar epithelial cells through WNT/β-catenin signaling pathway.This will provide a theoretical basis for studying the pathogenesis of BPD and provide a new therapeutic target for the early prevention and treatment of the disease.Methods:hyperoxia-induced neonatal rat BPD model was prepared according to the previous method^[1].Sprague-Dawley（SD）neonatal rats were randomly divided into two groups:model group（Fi O2=0.85）and control group（Fi O2=0.21）.Lung tissue samples were taken and primary type Ⅱ alveolar epithelial cells（AEC Ⅱ）were extracted at DAY 1,DAY 3,DAY 7,DAY 10 and DAY 14 after modeling.HE staining was used to verify the morphologic changes in lung tissue,and lung development was evaluated by Radial alveolar counts（RAC）.Western blot was used to detect the expression levels of TOP2A andβ-catenin in lung tissues of BPD model.The localization and expression of TOP2A in lung tissue of BPD model were verified by immunofluorescence.The extracted primary AEC Ⅱ was identified by immunofluorescence and the localization and expression of TOP2A in primary AEC Ⅱ of BPD model was verified;The protein and m RNA expression levels of TOP2A,β-catenin in primary AEC Ⅱ of BPD model were verified by q RCR and western blot techniques;The interaction between TOP2A andβ-catenin in BPD model was investigated by immunoprecipitation.The co-localization and expression of TOP2A andβ-catenin in primary AEC Ⅱ were further verified by immunofluorescence double staining.On the basis of the former methods,si RNA-TOP2A has been applied to interfere with primary AEC Ⅱ.western blot and immunofluorescence double staining techniques have been applied to proveβ-catenin and surfactant protein C（SFTPC）（marker of type Ⅱ alveolar epithelial cells）and aquaporin 5（AQP5）（marker of type I alveolar epithelial cells）.Results:HE staining and RAC values showed that there was no significant difference in lung morphology between the model group and the control group at DAY 3.At DAY 7,the model group began to show differences in lung tissue morphology,which showed that the number of alveoli decreased,the volume of alveoli increased,and the thickness of secondary septum increased.The gap between DAY 10 and DAY 14 model group and the control group was further enlarged,the number of alveoli decreased significantly,the volume of alveoli increased significantly,the size of alveoli was uneven,the shape was irregular,and the structure was simplified.Lung development in the model group lagged far behind that of the control group.At the tissue level,Western blot showed that the protein TOP2A expression in lung tissues of the BPD model had no significant difference at DAY 1 and DAY 3,but began to increase at DAY 7（P<0.01）,the expression continued to increase at DAY 10 and DAY 14（P<0.01）.While the protein expression levels ofβ-catenin were not significantly different at DAY 1 and DAY 3,but decreased at DAY 7（P<0.01）,DAY 10（P<0.01）and DAY 14（P<0.01）.Both tissue and cellular immunofluorescence showed that TOP2A was expressed in both the nucleus and cytoplasm,but mainly localized in the nucleus.With prolonged exposure time of hyperoxygen,TOP2A expression in the nucleus of AEC Ⅱ cells increased.At the primary AEC Ⅱ level,At the level of primary AEC Ⅱ,q PCR results showed that the expression of TOP2A m RNA in the model group was higher than that in the control group at DAY 7（P<0.01）,DAY 10（P<0.001）and DAY 14（P<0.001）,while the expression ofβ-catenin m RNA in the model group was lower than that in the control group at DAY 7（P<0.01）,day 10（P<0.001）and DAY 14（P<0.001）.The expression trend of TOP2A andβ-catenin was opposite.Western blot showed that the protein expression levels of TOP2A in the primary AEC Ⅱ of BPD model were higher in the model group than in the control group at DAY 7（P<0.05）,DAY 10（P<0.05）and DAY 14（P<0.01）.β-catenin expression levels were lower than in the control group at DAY 7（P<0.05）,DAY 10（P<0.05）and DAY 14（P<0.01）.The interaction between TOP2A andβ-catenin was demonstrated by immunoimmunoprecipitation in lung tissue of BPD model.The relationship between TOP2A andβ-Catenin was further explored.The co-expression of TOP2A andβ-Catenin was enhanced in the model group by cellular immunofluorescence.In vitro primary AEC Ⅱ experiments,western blot results showed that after TOP2A knockout,β-catenin expression was increased,AQP5 expression was decreased,and SFTPC expression was increased.Immunofluorescence double staining showed that the expression of SFTPC was increased and the expression of AQP5 was decreased after TOP2A knockdown.Conclusion:In the hyperoxia-induced neonatal rat BPD model,the expression level of TOP2A increased with prolonged exposure to high oxygen,and TOP2A directly interacted withβ-catenin in the WNT/β-catenin signaling pathway.The expression level ofβ-catenin decreased when the expression level of TOP2A was increased.After knockdown of TOP2A,the expression level ofβ-catenin increased,suggesting that TOP2A can inhibit the activation of WNT/β-catenin signaling pathway.TOP2A can promote the transdifferentiation of AEC Ⅱ to AEC Ⅰ by inhibiting the WNT/β-catenin signaling pathway.