The Role And Mechanism Of STUB1 Regulating PHLDA1 In The Pathogenesis Of COPD Emphysema Phenotype

Objectives: The emphysema phenotype is one of the clinical phenotypes of chronic obstructive pulmonary disease(COPD),and its morbidity and mortality are high.The lobular structure of lung tissue in patients with COPD emphysema phenotype is damaged,and in severe cases,there is chest tightness and shortness of breath with slight activity,which seriously affects the quality of life of patients.At present,there are no effective drugs for this phenotype,so it is important to find therapeutic targets to repair emphysematic lesions and prevent progression.Type II alveolar epithelial cell(AECII),also known as secretory cell,is a "stem cell" in lung tissue that plays a regenerative repair function,which plays an important role in the repair of alveolar epithelial cells.The number of ACEII is reduced,which will cause damage to the structure and function of alveolar tissue.PHLDA1(Pleckstrin Homology Like Domain Family A Member 1)belongs to the plateletleukokinase C substrate homologous(PH)domain family,which can directly or indirectly participate in the regulation of apoptosis through multiple pathways.Previous studies of the research group have shown that the apoptosis level of patients with emphysema phenotype is increased,and in vitro cell experiments have confirmed that PHLDA1 plays an anti-apoptotic role in regulating ACEII apoptosis.The protein expression level of PHLDA1 in COPD emphysema tissue was reduced compared with that in the control group.Based on the above studies,this study further explored the mechanism of PHLDA1 expression in COPD emphysema phenotype II alveolar epithelial cells through in vitro experiments and other methods.It provides a new strategy for the realization of targeted therapy for patients with emphysema phenotype.Methods:Part I:The expression of PHLDA1 in the COPD emphysema phenotype1.According to the clinical symptoms and pulmonary function examination results,eligible COPD patients are screened out.According to the chest CT image results,all subjects were divided into COPD non-emphysematous phenotype(COPD nonemphysematous phenotype)and COPD emphysematous phenotype(COPD emphysematous phenotype).2.Apply GEO(Gene Expression Omnibus database)database to screen differential expression genes for emphysema in chronic obstructive pulmonary disease.3.Immunofluorescence and immunohistochemistry kits were used to compare the localization and expression levels of PHLDA1 in lung tissues of patients with COPD nonemphysematic phenotype(COPD-NE)group and COPD emphysema phenotype(COPDE)group.4.Real-time quantitative polynucleotide chain reaction(rt-q PCR)and western blot(Western Blot)were used to detect the difference in the expression of PHLDA1 in the COPD-NE group and the COPD-E group.5.Different concentrations of cycloheximide(CHX)were added to the human type II alveolar epithelial cell line.We use CCK8 to detect the effect of different concentrations of cycloheximide on cell viability at different time points.After adding the optimal concentration of actinohelide,we use western blot to measure the protein expression of PHLDA1 at different time points.6.We use CCK8 to detect the cell viability at different time points after adding different concentrations of proteasome inhibitor Mg132.After adding the optimal concentration of Mg132,the protein expression of PHLDA1 is detected by Western Blot at the optimal time point.7.We use co-immunoprecipitation technology to detect the binding of PHLDA1 to ubiquitin in different groups of cells.Part II:E3 ligase STUB1 regulates PHLDA1 expression levels.1.We use co-immunoprecipitation to detect proteins interacting with PHLDA1 in human type II alveolar epithelial cell lines,which performs mass spectrometry analysis on the gel obtained by PHLDA1 co-immunoprecipitation.That obtains specific interaction protein profiles and completes its functional annotation,and finds genes that may be related to ubiquitination.2.We use the Ubi Browser database to predict the E3 ligase of PHLDA1 through bioinformation analysis.3.The expression difference of E3 ligase STUB1 in COPD-NE group and COPD-E group was verified by Western-Blot and tissue immunofluorescence.The expression levels of STUB1 in different groups of other type II alveolar epithelial cell lines were detected by Western-Blot.We use co-immunoprecipitation experiments to verify whether there was an interaction between the E3 ligase STUB1 and PHLDA1 in the human type II epithelial cell line.4.We respectively use Si RNA(small interfering RNA)transfection and p EX4 plasmid transfection down-regulated and up-regulated the expression level of STUB1 in cells.Knockdown and overexpression of STUB1 were followed by Western-Blot to detect the protein expression of PHLDA1.Part III:Nrf2 improves PHLDA1 expression by regulating PHLDA1 misfolding.1.We use immunofluorescence double staining technology to verify PHLDA1 misfolding of different groups of cells in human type II alveolar epithelial cell line.2.We use immunofluorescence double staining and Western-Blot to detect the differences of Nrf2 protein expression in different lung tissues.3.We add Nrf2 agonist t BHQ to different groups of human type II alveolar epithelial cell line cells.The protein expression of PHLDA1 was detected by Western-Blot and the co-immunoprecipitation experiment has verified the interaction about PHLDA1、Nrf2 and STUB1.4.PHLDA1 misfolding was detected by immunofluorescence double staining after adding Nrf2 agonist t BHQ and inhibitor ML385 to different groups of human type II alveolar epithelial cell lines.5.We use flow cytometry to verify apoptosis of different groups of human type II alveolar epithelial cell line.Results:Part I:The expression of PHLDA1 in the COPD emphysema phenotype.1.The results of GEO database analysis show that PHLDA1 is a differentially expressed gene between COPD-E group and COPD-NE group.2.The results of Western Blot,immunohistochemistry and immunofluorescence showed that PHLDA1 was low in lung tissues of patients in COPD-E group compared with COPD-NE group,mainly expressed in type II alveolar epithelial cells of human lung tissue,and mainly localized in the cytoplasm.3.The results of Rt-q PCR showed that PHLDA1 expression level in lung tissue of patients in COPD-E group was increased compared with that in COPD-NE group,which was opposite to that of protein.4.The results of actinomycone experiment showed that the degradation of PHLDA1 in the cigarette smoke extract(CSE)group was increased compared with the control group,and the protein half-life was shorter.5.The protein expression of PHLDA1 in CSE group increased significantly compared with CSE group after adding proteasome inhibitor Mg132 to human type II epithelial cell line compared with CSE group alone.6.The results of co-immunoprecipitation experiments showed that the degree of ubiquitination of PHLDA1 in C SE group was significantly higher than that in control group.Part II: E3 ligase STUB1 regulates protein expression levels of PHLDA11.Co-immunoprecipitation and mass spectrometry analysis of genes associated with PHLDA1 in the human type II alveolar epithelial cell line yielded 2902 genes interacting with PHLDA1 and 197 genes that may be related to ubiquitination.2.There are 35 E3 ligases of PHLDA1 predicted by bioinformation analysis.STUB1 is a gene shared in mass spectrometry and bioinformatics analysis.STUB1 is presumed to be the E3 ligase of PHLDA1.3.The results of immunofluorescence double staining of human lung tissue showed that STUB1 and PHLDA1 could be colocalized,and STUB1 was highly expressed in lung tissues of patients in COPD-E group.4.The results of Western Blot showed that STUB1 was highly expressed in lung tissue of patients in COPD-E group compared with COPD-NE group.The expression of STUB1 in C SE group was higher in human type II epithelial cell line than in control group.5.The results of co-immunoprecipitation experiments showed that there was an interaction between STUB1 and PHLDA1 in human type II epithelial cell lines.6.The protein expression of PHLDA1 increases after knocking down STUB1,and the protein expression of PHLDA1 decreases after overexpressing STUB1.Part III: Nrf2 improves PHLDA1 protein expression by regulating PHLDA1misfolding1.The results of immunofluorescence double staining showed that PHLDA1 was misfolded,and the CSE group was more misfolded than PHLDA1 compared with the control group.2.Immunofluorescence double staining of human lung tissues confirmed that the expression of Nrf2 in lung tissues of patients in the COPD-E group was reduced,and PHLDA1 and Nrf2 could be colocalized.Western Blot detected a decrease in the expression of Nrf2 in the COPD-E group.In human type II alveolar epithelial cells,Western Blot results showed a decrease in the expression of Nrf2 in CSE group cells.3.The addition of Nrf2 agonist t BHQ to human type II alveolar epithelial cells,and the addition of 6% CSE after 24 h,Western Blot results showed that the CSE group with t BHQ added increased protein expression compared with PHLDA1 in the CSE group alone.4.The results of co-immunoprecipitation showed that Nrf2,P HLDA1 and STUB1 could interact.5.After the addition of Nrf2 agonist t BHQ and inhibitor ML385 in the CSE group,the results of immunofluorescence double staining showed that the misfolding of PHLDA1 in the ML385 group was increased compared with that in the t BHQ group.6.The apoptosis of different groups of cells was detected by flow cytometry,and the results showed that t BHQ could improve the apoptosis rate of human type II alveolar epithelial cells.Conclusion:1 PHLDA1 is mainly expressed on human type II alveolar epithelial cells.2.The expression of PHLDA1 protein in COPD-E group decreased compared with that in COPD-NE group.3.The half-life of PHLDA1 protein in CSE group becomes shorter,and it is mainly degraded by the ubiquitin-proteasome pathway.4.STUB1 is the E3 ligase of PHLDA1,which regulates the protein expression of PHLDA1.5.Cigarette smoke extract(CSE)leads to increased PHLDA1 misfolding.6.Upregulating Nrf2 reduces PHLDA1 misfolding and reduces apoptosis.7.PHLDA1、STUB1、Nrf2 may become new targets for the phenotypic treatment of COPD emphysema.