The Effects And Regulatory Mechanisms Of TROP2 In Airway Basal Cells

BackgroundAirway basal cells(BCs)are located in the bottom of airway pseudostratified epithelium,which are cubic in shape and closely attached to the basement membrane.In 1991,Randell et al.successfully isolated airway BCs by flow cytometry,and proved that BCs could self replicate and differentiate into ciliated columnar cells and mucus cells.It is first proposed that BCs may be airway stem cells.After nearly 30 years of unremitting efforts,scientists have gradually reached a consensus:Airway BCs are generally accepted as a stem/progenitor cell population.In recent years,great progress has been made in the biological research of human airway BCs,including the characteristics of gene expression,stem/progenitor cells and other functions,as well as the interaction with other types of airway cells.BCs can not only influence peripheral cells by secreting polypeptide substances,but also receive the stimulation of peripheral cells and respond through various receptors.Therefore,BCs play a central role in the regulation of airway homeostasis.Abnormal number or function of BCs can lead to disruption of airway epithelial homeostasis.Excessive proliferation will lead to cell proliferation;abnormal differentiation can lead to goblet cells hyperplasia or squamous metaplasia.These changes are closely related to the pathological manifestations in COPD.At the 2016 American Thoracic Society International Conference(ATS),Professor Shaykhiew,together with Professor Petracher hypothesized that airway BC is a primary driver of COPD pathogenesis.Therefore,a better understanding of the function and regulatory mechanism of BCs has important clinical significance.Recently,we carried out several studies to determine the distribution and function of trophoblast antigen protein 2(TROP2)in adult lung.TROP2 is a transmembrane glycoprotein encoded by the TACSTD2 gene.Previous studies have mainly focused on abnormal expression of TROP2 in cancer cells.Recent studies reveal that TROP2 promotes stem cell-like qualities in cells and regulates cell growth,transformation,regeneration,and proliferation.Our preliminary results in adult lungs indicate that TROP2 is mainly distributed in BCs,and is up-regulated in the epithelium of COPD.This up-regulation of TROP2 may affect BC function,specifically by inducing BC hyperplasia and EMT-like changes,and causing the release of inflammatory molecules into the microenvironment.These changes coincide with the pathological features of COPD,and thus present solid evidence that TROP2-mediated reprogramming of airway BC phenotype and function play a role in COPD-associated abnormal airway homeostasis.With the deepening of research,more and more questions attract us to explore:what is the function of TROP2 on airway BCs,and what roles it may play?What is the underlying mechanism of TROP2 expression?In view of the above questions,the present stdudy mainly focuses on the following four aspects:1.Histological study on TROP2 expression in airway BCs of COPD patients;2.Proteomic analysis on BCs with high expression of TROP2;3.Regulation of LOXL2 by TROP2 and its effect on biological behavior of BCs;4.Regulatory mechanism of TROP2 expression and intervention of ginsenoside Rb3.1.Histological study on TROP2 expression in airway BCs of COPD patients1.1 Objective:To investigate TROP2 expression in lung tissues of COPD patients,and to analyze the correlation with lung function,airway remodeling indicators and smoking index,so as to provide pathological basis for analyzing the effect of TROP2 on BC function.1.2 Materials and methods:(1)Participants:All protocols,including recruitment of participants,collection of tissue samples and analysis of clinical data,were approved by the ethics committee of Qilu Hospital,Shandong University(KYLL-2018-316).Patients who underwent lobectomy for lung diseases from 2018 to 2020 were recruited(2)Clinical data were collected,including:general data:gender,age,smoking index,past medical history,current medical history,etc.;pulmonary function data,including FEV1%predicted and FEV1/FVC.(3)Collection of lung tissue specimens:lung tissues containing grade 3-4 bronchial stump and peripheral lung tissue specimens were simultaneously collected,fixed in formalin,embedded in paraffin,and serial sectioned(4)The TROP2 expression in large and small airway epithelium was detected by immunohistochemistry.(5)Immunofluorescence was used to detect the location of TROP2 in airway epithelium.(6)Sirius red was used to detect the collagen content of airway basement membrane.(7)TROP2 expression difference between groups and corresponding correlation analysis were conducted.1.3 Results(1)Clinical characteristics of participants:The present study included COPD group(32 cases),smoking control group(46 cases)and non-smoking control group(50 cases),and there was no significant difference in age among the groups.(2)Immunofluorescence showed that TROP2 and KRT-5 were co-stained in the both large and small airway epithelium,which confirmed that TROP2 protein was mainly expressed in airway BCs.(3)TROP2 expression in airway BCs of COPD patients was significantly higher than that in smokers or non-smokers with normal lung function.Correlation analysis showed that the TROP2 expression was negatively correlated with FEV1%and FEV1/FVC.(4)In COPD group,the deposition of collagen in the basement membrane of large airway was increased,which was positively correlated with TROP2 expression.(5)TROP2 expression was positively correlated with smoking index in smokers.1.4 Summary(1)TROP2 is mainly expressed in BCs in the airway epithelium.(2)The TROP2 expression in airway BCs of COPD group was significantly higher than that of control group,and was positively correlated with the degree of airflow obstruction and the content of collagen in the basement membrane.(3)The TROP2 expression in airway BCs of smokers was higher than that of non-smokers,and was positively correlated with smoking index.2.Proteomic analysis on TROP2 overexpression BCs2.1 Objective:In this part,we constructed a BC model with stable and high expression of TROP2,performed proteomic analysis,and verificated the corresponding differential proteins,so as to provide clues for further exploring the dysfunction of BCs mediated by TROP2.2.2 Materials and methods:(1)Culture and identification of primary airway BCs:The patients with unilateral localized peripheral lung nodules were selected for routine electronic bronchoscopy examination.They had no history of smoking,with normal pulmonary ventilation function.The bronchial epithelium was gently brushed with sterile brush,and the BCs were cultured and expanded with BEGM.The BCs were identified by p63 and KRT-5.(2)Construction,purification and identification of BC model with upregulated TROP2 expression①Construction of lentivirus:Constructions of TROP2 lentivirus vector and control sequence plasmid(5NC)from Invitrogen Company were entrusted.②293T cells were cotransfected with RNAi mate.The supernatant rich in lentivirus particles was collected and concentrated to obtain high titer lentivirus concentrate.③The lentivirus transfection experiment of BCs:TROP2 overexpression group,lentivirus transfection control group and blank control group were set.④Purinomycin was used to screen the purity of TROP2 high expression BCs.⑤Western blot was used to identify the BCs with high expression of TROP2.(3)Whole solution enzymolysis and nanoupgrading reverse phase chromatography Orbitrap fusion for protein analysis①Extraction,quantification and quality control of total protein:TRIZOL method was used to extract total protein,and Bradford method was used to determine the concentration of extracted protein.②Mass spectrometry data processing:Database:UniProt Human(2019.4.20 download)database.The mass spectrum analysis of Label free is completed by thermo Orbitrap fusion mass spectrum,and the original mass spectrum file is processed by Maxquant software③Overall analysis and differential protein screening:Maxquant significance was used to test the significance of the difference,with P value of 0.05 and fold change of 1.2 times.④Verification of differential proteins:Western blot,ELISA and Immunofluorescence were used to verify the proteomic results.⑤Function analysis:GO and KEGG analysis.2.3.Results(1)Primary culture and identification of airway BCs:airway BCs were obtained by bronchoscopic brushing and BEGM culture.Immunofluorescence staining showed that all cells expressed BC markers KRT-5 and p63 at the same time.(2)Construction of lentiviral vector:Homo-TROP2:1.8 x 1010tu/ml,lv5-NC:1.0 x 109tu/ml.The transfection efficiency was detected by Western blot:TROP2 expression protein in the high expression group was 2 times higher than that in the control group.(3)Protein identification results:a total of 23658 peptide fragments were identified from the corresponding 2718 proteins.(4)Protein difference analysis:17 differential proteins were obtained,of which 15 proteins were up-regulated and 2 proteins were down regulated.These include:up regulation of AMPS,DCD and S100A7,up regulation of MUC5AC and MUC5B,up regulation of LOXL2 and tenascin C,up regulation of integrin subunits including integrin a V,integrin α 2 and integrin α 2.The expression of SMC2 and NUDC increased,but the expression of Cep 164 decreased.(5)Verification of differentially expressed proteins①Western blot showed that both MUC5AC and MUC5B were upregualted in TROP2 highly expressed BCs.②Western blot and immunofluorescence showed that LOXL2 was upregualted in TROP2 highly expressed BCs.③ELIS A showed that dermcidin and S100A7 in the supernatant of basal cell culture medium with high expression of TROP2 was up-regulated compared with the control group.(5)Function annotation①Go analysis:the differentially expressed proteins of TROP2 were mainly located in the cell membrane.These differential proteins mainly affect molecular biological functions,such as protein glycosylation,cell division,mitochondrial translation and elongation,defense response of Gram-negative bacteria,regulation of T cell-mediated cytotoxicity,cell adhesion and so on.②KEGG analysis:the differentially expressed proteins were significantly enriched in viral infection and immune response pathway,RIg-Ⅰ receptor signaling pathway,apoptosis,MAPK signaling pathway,breast cancer and small cell lung cancer pathway.2.4 Summary(1)The differential proteins elicited by TROP2 overexpression in BCs involve innate immune function,proliferation and migration ability,and extracellular matrix regulation.(2)The expression of LOXL2 in the basal cell model with TROP2 high expression was dramatic upregulated,which may play an important role in the abnormal biological behavior of BCs mediated by TROP2.3.Regulation of LOXL2 by TROP2 and its effect on biological behavior of BCs3.1 Objective:To verify the correlation between LOXL2 and TROP2 at the histological and cytological levels,and to explore the role of LOXL2 in basal cells with high expression of TROP2.3.2 Materials and methods(1)LOXL2 epxression in Lung tissue samples:The expression of LOXL2 in airway epithelium of COPD and control group was detected by immunohistochemistry,and the correlation with lung function parameters,basement membrane collagen content and TROP2 expression level was analyzed.(2)The effect of TROP2 on LOXL2 expression:the stable high/low expression BC models of TROP2 were constructed to detect the effect of TROP2 expression on LOXL2.(3)Effect of LOXL2 up-regulation on BC function:β-aminopropionitrile(BAPN)is the most commonly used LOXL2 inhibitor.CCK-8,scratch test,invasion test and Western blot were used to evaluate the proliferation,repair,migration and EMT of BCs with TROP2 high expression.(4)Regulatory mechanism of TROP2 on LOXL2:Regulation of LOXL2 expression by NEMO/NF-κB pathway was further studied by Western blot.3.3.Results(1)LOXL2 expression was up-regulated in the airway epithelium of COPD:Immunohistochemical results showed that the expression of LOXL2 was up-regulated in the airway epithelium of COPD,and positively correlated with TROP2 expression and the content of basement membrane collagen,and negatively correlated with FEV1%and FEV1/FVC.(2)Effect of TROP2 on LOXL2 expression:high expression of TROP2 up-regulated LOXL2 expression,while interference of TROP2 inhibited LOXL2 expression.(3)TROP2 can regulate the activity of NF-κB pathway through NEMO:high expression of TROP2 can up regulate NEMO and activate NF-κB pathway,while interference of TROP2 can down regulate NEMO expression and inhibit the activation of NF-κB pathway(4)In BCs with high expression of TROP2,the LOXL2 expression was down regulated by inhibiting NEMO and NF-κB pathway respectively.(5)The effect of up-regulation of LOXL2 by TROP2 on the biological behaviors of BCs:the proliferation,migration and wound healing ability of BCs with overexpression of TROP2 were significantly enhanced,and significant EMT changes were also observed,while the above changes were partially inhibited by the addition of LOXL2 inhibitor.3.4 Summary(1)LOXL2 was up-regulated in the airway epithelium of COPD,and was positively correlated with TROP2 expression.(2)TROP2 up-regulates the LOXL2 expression through NEMO/NF-κB pathway,thereby enhancing the proliferation and migration of basal cells and inducing EMT changes in basal cells.4.Regulatory mechanism of TROP2 expression and intervention of ginsenoside Rb34.1 Objective:In this part,we observed the effect of cigarette extract(CSE)on the expression of TROP2 in basal cells,analyzed the possible molecular mechanism,and explored the regulatory effect of ginsenoside RB3 on the expression of TROP2.4.2 Materials and methods(1)Observe the effect of CSE stimulation on TROP2 expression in BCs:the BCs were stimulated with gradient concentration CSE(final concentration:0,1%,2%,4%and 8%)for 48 hours,or at specific time intervals(0 h,6 h,12 h,24 h,48 h and 72 h)with 4%CSE.Western blot was used to detect TROP2 expression in BCs.The activation of p38 MAPK pathway and NF-κB pathway were also evaluated.(2)Investigate the effect of p38 MAPK pathway and NF-κB pathway on TROP2 expression:SB203580 and PD169316 are commonly used small molecule inhibitors of p38 MAPK pathway.Two different concentrations(SB203580,final concentration 10μM/20μM;PD169316,0.1μM/1μM)were added with each inhibitor for 48 h.TROP2 expression in BCs was detected by Western blot.NF-κB pathway specific inhibitor(BAY 11-7082)and siRNA knockout p65 were used to inhibit NF-κB pathway.(3)The effect of ginsenoside Rb3 on TROP2 expression in BCs was observed in monolayer culture:1 hour before the addition of 4%CSE,different concentrations of ginsenoside Rb3(5μM,10μM,20μM and 50μM)were added to the culture medium respectively.After 48 hours of stimulation,TROP2 expression and the activity of p38 MAPK and NF-κB pathway were detected by Western blot.(4)Verify the regulatory effect of ginsenoside Rb3 on TROP2 expression in human airway BCs by air-liquid interface culture(ALI)model①The ALI culture model was established according to brekman et al.From the first day,CSE stimulation and/or 5μM,10μM ginsenoside Rb3 were added in the lower chamber.②On the 28th day,the model of ALI was evaluated by hematoxylin eosin staining and immunofluorescence.③ Western blot and immunofluorescence were used to detect TROP2 expression protein,and the activation of p38 MAPK pathway and NF-κB pathway was evaluated.④Western blot was used to detect the expression of LOXL2 and EMT markers while immunofluorescence was used to evaluate the proliferation of BCs.⑤On the 14th and 28th day,the supernatant of ALI medium was collected for ELISA to dectect inflammatory factors4.3 Results(1)CSE induces TROP2 expression in airway BCs:CSE stimulated TROP2 mRNA and protein expression in BCs in a dose-dependent and time-dependent manner(2)CSE induces p38 MAPK and NF-κB pathway activation:CSE activated p38 MAPK and NF-κB pathways in a concentration and time-dependent manner.(3)Antagonists of p38 MAPK pathway inhibit TROP2 expression in BCs:PD 169316 or SB203580 could significantly inhibit the up regulation of TROP2 induced by CSE.(4)Down-regulation of NF-κB pathway inhibits TROP2 expression in BCs:NF-kB pathway inhibition by BAY11-7082 or siRNA transfection inhibits TROP2 expression in BCs.(5)Ginsenoside Rb3 inhibits TROP2 expression,p38 MAPK and NF-κB pathway in monolayer culture:Western blot analysis showed that ginsenoside Rb3 at a low concentration(5 μM)inhibited TROP2 expression protein induced by CSE,and increasing the concentration of ginsenoside Rb3 could enhance its ability to inhibit TROP2 expression.The induction of pp38 and pp65 by CSE was inhibited by ginsenoside Rb3(6)Ginsenoside Rb3 antagonized CSE induced up regulation of TROP2 in ALI culture TROP2 expression in 4%CSE group increased by about 2 times.After adding 10 μm ginsenoside Rb3，TROP2 expression was down regulated to baseline level,and the levels of pp3 8 and pp65 were also decreased.(7)Ginsenoside Rb3 antagonized the proliferation,inflammatory response and EMT changes of BC induced by CSE.①The positive rate of Ki67 was significantly increased in 4%CSE group.The Ki67 index decreased significantly after adding 5μM or 10μM ginsenoside Rb3.Correspondingly,frequency of p63+BCs increased significantly in the CSE stimulated group,while decreased to near normal level when 5 μM or 10 μM ginsenoside Rb3 was added to CSE.②Rb3 significantly down-regulated Vimentin and increased E-cadherin level,which indicated that the change of EMT induced by CSE could be reversed by ginsenoside Rb3.③Rb3 significantly down-regulated CSE induced LOXL2 expression.④Compared with the control group,the levels of three factors(IL-6,IL-8 and IL-1β)in CSE stimulation group were increased.5 μM or 10 μM ginsenoside Rb3 inhibited the secretion of IL-6,IL-8 and IL-1β.4.4 Summary(1)Cigarette smoke extract induces the expression of TROP2 in basal cells through p38 MAPK and NF-κB pathway.(2)Ginsenoside Rb3 inhibits the expression of TROP2 induced by CSE by antagonizing p38 MAPK and NF-κB pathway,and antagonizes the dysfunction of basal cells induced by CSEConclusions(1)The differential proteins elicited by TROP2 overexpression in BCs involve innate immune function,proliferation and migration ability,and extracellular matrix regulation.(2)TROP2 promotes the proliferation,migration and EMT changes of BCs by regulating LOXL2 expression.(3)Ginsenoside Rb3 inhibited CSE-induced TROP2 expression and associated dysfunction of BCs by blocking p38 MAPK and NF-κB pathways.