The Screening Of Serum Protein Biomarkers For Biliary Atresia And The Role Of Polymeric Immunoglobulin Receptor In The Pathogenesis Of Biliary Atresia

Objective: Biliary atresia(BA)is a fatal neonatal cholangiopathy characterized by progressive bile duct injury and liver fibrosis.The pathogenesis of BA has not yet been elucidated,and the diagnosis and management of BA are currently challenging.In this study,we aimed at discovering new serum biomarkers for BA diagnosis,and investigating the role of polymeric immunoglobulin receptor(PIGR)in BA pathogenesis.Methods:1.Part Ⅰ:Data-independent acquisition mass spectrometry(DIA-MS)was applied to analyze the protein expression profile in the serum samples of 11 BA and 9age-matched healthy control patients.The proteome data was in-depth analyzed to reveal the expression feature of BA,and to find candidate BA biomarkers.Ten candidate serum biomarkers were selected and validated in an independent patient cohort of 50 BA,56non-BA cholestasis(CS)and 30 healthy controls(HC)using enzyme-linked immunosorbent assay(ELISA).The diagnostic value of biomarkers was assessed with receiver operating characteristic curve(ROC)by calculating the area under ROC(AUC).The validated biomarkers were combined with clinical indexes of BA to further investigate their diagnostic potential.2.Part Ⅱ:PIGR expression was detected in the liver and extrahepatic bile ducts(EBD)biopsies of BA and control patients by using quantitative PCR and immunofluorescence staining.Rhesus rotavirus(RRV)induced BA mouse model was conducted,and PIGR expression was detected using quantitative PCR,western blot and immunofluorescence staining.Human intrahepatic biliary epithelial cells(Hi-BEC)were cultured in vitro,supplied with different recombinant human cytokines,to find the stimulators of PIGR up-regulation.Ammonium pyrrolidinedithiocarbamate(PDTC),an inhibitor of nuclear factor kappa B(NF-κB)signaling,was applied to investigate the association between NF-κB signaling and cytokine-induced PIGR upregulation.Small interferon RNA(si RNA)and lentiviral expression vector were used for PIGR silence and overexpression in the Hi-BEC.The proliferation of Hi-BEC was measured by cell counting kit 8(CCK8)assay,and the apoptosis of Hi-BEC was analyzed using flow cytometric and TDT-mediated d-UTP Nick-End Labeling(TUNEL).The epithelial-mesenchymal transition of Hi-BEC was measured by detecting the expression levels of actin alpha(α-SMA)and E-cadherin.Results: 1.Part Ⅰ: The serum proteome analysis revealed distinct features of BA,such as abnormal metabolism of oxoacid,organic acid,carboxylic acid and nuclear acid,deficiency in embryo development and multicellular organism development,and dysregulation of immune response and inflammation.Ten candidate proteins,with an AUC higher than 0.9 and upregulated in BA,which are alpha fetoprotein(AFP),polymeric immunoglobulin receptor(PIGR),intercellular adhesion molecule 1(ICAM1),aldolase,fructose-bisphosphate B(ALDOB),cathepsin D(CTSD),poliovirus receptor(PVR),fumarylacetoacetate hydrolase(FAH),HECT and RLD domain containing E3 ubiquitin protein ligase 2(HERC2),apolipoprotein E(APOE),and fibronectin type Ⅲ and SPRY domain containing 1(FSD1),were selected as candidate biomarkers for further analyses.The ELISA result showed that all candidate biomarkers were upregulated in BA serum,compared with healthy controls,and presented high diagnostic accuracy in distinguishing BA from HC;however,only ALDOB,PIGR and PVR presented satisfactory(with an AUC higher than 0.7)accuracy in distinguishing BA from non-BA cholestasis.ALDOB,PIGR and PVR showed higher AUC than gamma-glutamyl transpeptidase(GGT),and reached a higher AUC when they combined with GGT.The in combination of PIGR and PVR showed highest diagnostic accuracy with an AUC of0.911.In BA patients,serum PVR level correlated with GGT level,ALDOB level correlated with liver fibrosis,and PIGR correlated with both liver fibrosis and liver elasticity.2.Part Ⅱ: PIGR m RNA was significantly upregulated in the liver and EBD tissue of BA;and in the liver tissues,the PIGR antigen was located in the bile ducts epithelium.In RRV-induced BA models,PIGR was upregulated in the liver at D7 and D14(post-infection),and located in the bile ducts epithelium.In cultured Hi-BEC,recombinant human IL-1β,TNF-α,IFN-γ and IL-17 A triggered the upregulation of PIGR;inhibiting the activation of NF-κB restrained the IL-1β,TNF-α and IFN-γ induced PIGR expression,and partially reduced the expression level of IL-17 A induced PIGR expression.In BA livers,PIGR m RNA expression level positively correlated with both liver IL-17 A m RNA expression level and serum PIGR protein level.Silencing PIGR suppressed the proliferation,and increased the apoptosis of Hi-BEC;while overexpressing PIGR promoted proliferation and decreased the apoptosis of Hi-BEC.PIGR overexpressed Hi-BEC upregulated the expression of α-SMA and downregulated the expression of N-cadherin.Conclusion: 1.ALDOB,PIGR and PVR increased in the serum of BA patients,and could be used as diagnostic biomarkers of BA.2.PIGR was upregulated in the liver of BA patients,located in biliary epithelium,and PIGR expression significantly correlated with IL-17 A expression level.3.In cultured Hi-BEC,recombinant human IL-1β,TNF-α,IFN-γ triggers the upregulation of PIGR in a NF-κB dependent manner,while IL-17 A induced PIGR expression in both NF-κB dependent and NF-κB independent signaling pathways.4.PIGR participates BA pathogenesis via regulating the proliferation,apoptosis,and EMT process of cholangiocytes.