Mechanism Of S100A6 In Intrahepatic Bile Duct Epithelial Cells Injury In Primary Biliary Cholangitis

Objective: Primary biliary cholangitis(PBC)is a kind of chronic intrahepatic cholestasis autoimmune disease involving intrahepatic interlobular bile ducts.PBC can occur at all ages,mostly from 30 to 70 years old,with the peak at 50 years old.It is more common in women than in men,with a male to female ratio of 1: 9-10,and it is the most common reason for women to undergo liver transplantation.Patients with PBC often have symptoms that affect quality of life,such as fatigue and intractable itching.A total of 30%of patients with PBC may develop decompensated liver disease or die.With the development of serum autoantibody detection and the improvement of Chinese people’s attention to liver diseases,the number of patients with PBC detectable in China is also on the rise.At present,there is no large-scale epidemiological investigation in China.An epidemiological survey in Hong Kong,China,found that the incidence of PBC reached82.3 per million in 2015.The main pathological features of PBC are that the immune system specifically recognizes and kills intrahepatic bile duct epithelial cells,leading to the gradual disappearance of intrahepatic bile ducts and cholestasis.With the progress of the disease course,liver tissue is regenerated repeatedly and gradually fibrosis,eventually leading to liver failure.The Gene Expression Omnibus(GEO)is a widely used international public repsository of high-throughput functional genomic data.Bioinformatics analysis of existing microarray data in GEO database can be used to screen the differential genes in PBC liver tissue or cholestasis bile duct epithelial cells,so as to find the target genes of PBC.And the bioinformatics prediction can be verified by experimental means.Biliary duct ligation(BDL)is a common procedure for biliary obstruction and is widely used in rodent models of cholestasis and liver injury.By using BDL mouse model to simulate the occurrence of PBC disease,we can dynamically observe the damage of liver and intrahepatic bile duct epithelial cells,and verify whether there are differences in the expression of the screened target genes in intrahepatic bile duct epithelial cells.Intrahepatic biliary epithelial cells(i BECs)are an important component of hepatocytes,and their injury mechanism is the key to the study of the pathogenesis of PBC.The accumulation of bile salts as "cytotoxins" in liver tissue plays a key role in the development of hepatocyte necrosis and fibrosis.In this study,glycochenodeoxycholic acid(GCDC)was used to deal with human intrahepatic bile duct epithelial cells to simulate the PBC intrahepatic cholestasis environment,and the role of target genes in the injury mechanism of PBC intrahepatic bile duct epithelial cells was studied in vitro.A number of studies have shown that transcription factors are closely related to the occurrence and progression of diseases,and play an important role in regulating gene expression.In this study,bioinformatics methods were used to predict the transcription factor of target gene promoter,and to verify the transcription factor regulation of target gene promoter by experiments in vitro,and then to explore the intrahepatic bile duct cell damage mechanism of PBC.Long non-coding RNA(lnc RNAs)are involved in the regulation of intracellular process.A variety of lnc RNAs,as a structural component,can form the nucleic acid protein complex with transcription factors,as well as combined with specific transcription factor,which can change their cellular localization and affect the transcription of target genes.Abnormal expression of lnc RNAs in plasma have been shown to predict a variety of human diseases.In conclusion,animal model was used to verify the target genes predicted by bioinformatics.At the same time,intrahepatic biliary epithelial cells were used as the research object,and the regulation effect of transcription factors on target gene promoter was explored.The role of biliary epithelial cells injury induced by bile acids in the pathogenesis of PBC was illustrated.Target genes as biomarkers in plasma of patients were analyzed to evaluate the value of diagnosis and staging in PBC to provide the basis for disease monitoring and targeted therapy of PBC pathogenesis.Subjects: Bioinformatics analysis included data from GSE79850 and GSE29776 chips in the GEO database.Mouse model: 21 male C57BL/6J SPF mice aged 6-8 weeks.A total of 145 PBC patients diagnosed in the First Affiliated Hospital of China Medical University and 110 healthy subjects in the physical examination center were selected.The training set included 80 PBC patients and 60 healthy controls.The validation set included 65 PBC patients and 50 healthy controls.Cell lines: human intrahepatic biliay epithelial cell line;293T cell line.Methods:1.Bioinformatics analysis(1)Genespring software was used to analyze differential genes analysis of liver tissue in GSE79850 chip data.Genome ontology,pathway enrichment analysis and protein-protein interaction were analzyed,and literature was mined online.The key genes and transcription factors of PBC were analyzed.(2)The differentially expressed genes of GSE29776 chip BDL group and sham group were analyzed.(3)Differentially expressed genes were screened and verified by q RT-PCR in plasma of PBC patients and healthy controls.2.Validation of target gene in BDL mouse model(1)Establishment of BDL mouse model: experimental group(BDL)were treated with common bile duct ligation;the sham group mice received free common bile duct(suture)after laparotomy.The mice were sacrificed for liver tissue at 5,10 and 15 days after operation.HE and Masson staining were performed to observe the degree of liver tissue fibrosis,and the optimal modeling time was selected.(2)CK19 and S100a6 proteins in intrahepatic bile duct epithelial cells of BDL mice were detected by double immunofluorescence labeling to verify the bioinformatics prediction.3.Biological function of S100A6 in Hi BECs treated by GCDC(1)Screening of time and concentration of Hi BECs treated with GCDC: Hi BECs were treated with 200μm,500μm and 1000μm GCDC,respectively.The percentage of apoptosis was detected by flow cytometry after 2h,6h and 24 h culture,respectively.The concentration and culture time with greatest statistical difference were selected as the condition for subsequent experiments.(2)Immunohistochemistry was used to identify Hi BECs.(3)q RT-PCR and Western Blotting were used to detect the interference efficiency of S100A6 overexpressed plasmid and Smart silencer transfected Hi BECs.(4)The effect of S100A6 on the apoptotic ability of Hi BECs was detected by flow apoptosis.(5)CCK8 assay was used to detect the effect of S100A6 on proliferation of Hi BECs.4.Regulation of transcription factor ESR1 on S100A6 promoter(1)q RT-PCR and Western Blotting were used to detect the interference efficiency of ESR1 overexpressed plasmid and ESR1 Smart silencer transfected Hi BECs.(2)The effect of ESR1 on S100A6 m RNA expression was detected by q RT-PCR.(3)Western Blotting was used to detect the effect of transcription factor ESR1 on the expression of S100A6 and PDC-E2 proteins in Hi BECs.(4)The activation of S100A6 promoter by transcription factor ESR1 was verified by double luciferase reporter gene assay;(5)Hi BECs overexpressing ESR1 were transfected with S100A6 interference plasmid,and the apoptosis level of Hi BECs was detected by flow cytometry;and the proliferation of Hi BECs was detected by CCK8 assay.5.The value of plasma S100A6 and related lnc RNAs as biomarkers in the diagnosis and staging of PBC(1)Bioinformatics methods were used to predict lnc RNAs related to S100A6.(2)The relative expression levels of S100A6 m RNA and lnc RNAs in plasma of PBC patients and healthy controls were analyzed by q RT-PCR.(3)The value of S100A6 m RNA and related lnc RNAs in the diagnosis and staging of PBC was evaluated by ROC curves.Results:1.Bioinformatics analysis results(1)Bioinformatics analysis of liver tissue in patients with PBC(a)The gene expression profile of PBC patients was compared with that of the control group.A total of 77 DEGs(P < 0.05 and FC > 2.0),including 47 up-regulated genes and30 down-regulated genes;(b)GO enrichment analysis showed that all DEGs were significantly enriched in biological processes.The upregulated DEGs were mainly concentrated in immune response,cytokine response and cytokine stimulation response.(c)Pathway analysis: The up-regulated DEGs were mainly concentrated in influenza A and herpes simplex virus infection pathways.The down-regulated DEGs were mainly enriched in tumor necrosis factor(TNF)signaling pathway and NF-k B signaling pathway.(d)PPI score > 0.4 was selected for STRING analysis;74 nodes and 356 protein pairs were obtained;cluster analysis obtained two MCODE clusters(scores > 3);(e)Literature mining was conducted for genes with scores greater than 10 in the PPI network,and 27 previously reported genes enriched in cellular activation of innate immune response and immune system were constructed as co-citation networks.(f)Three key PBC-related genes listed in CTD were found in DEGS,including CCL5,IL7 R and TNFRSF1 A.At the same time,five transcription factors were identified and constituted a transcriptional regulatory network.(2)Differential gene screening of biliary epithelial cells in BDL mouse model: q RT-PCR was used to screen target genes in 30 PBC patients and 30 healthy controls.S100A6 in plasma of PBC patients had the greatest change(t = 20.28,P < 0.0001).2.Establishment of BDL mouse model and identification of differential genes(1)The liver histomathology of BDL group was consistent with liver fibrosis.(2)HE staining: There was significant difference in the number of inflammatory cells in portal area of liver tissue between BDL group and sham group(P < 0.05).(3)Masson staining: It can be clearly observed that the order of the portal area surrounding Ι type collagen fibrosis fiber,forming fiber intervals,interfibrous septa with lobular disorder,liver cirrhosis;the liver tissue in the sham group was well developed and unfibroable.(4)Double immunofluorescence labeling results showed that CK19 and S100a6 proteins had positive expression in the BDL mouse intrahepatic bile duct epithelial cells,and the two kinds of proteins were only weakly expressed in the sham group.3.S100A6 promotes apoptosis and inhibits proliferation of Hi BECs(1)Compared with NC group,expression level of S100A6 in Hi BECs was significantly increased after GCDC treatment(P < 0.05).(2)Compared with NC group,after transfection with S100A6 Smart silencer,it was showed that the expression of PDC-E2 protein in Hi BECs was decreased by Western Blotting(P < 0.05).After transfection with S100A6 overexpressed plasmid,the expression of PDC-E2 protein in Hi BECs was significantly increased(P < 0.05).(3)Compared with the NC group,the results of CCK8 assay showed that the proliferation of Hi BECs was significantly increased after transfection of S100A6 Smart silencer(P < 0.05).The Hi BECs proliferation was significantly reduced after transfection with S100A6 overexpressed plasmid(P < 0.05).(4)Compared with NC group,after transfection with S100A6 Smart silencer,the apoptosis of Hi BECs was significantly decreased by flow cytometric(P < 0.05);After transfection with S100A6 overexpressing plasmid,apoptosis level of Hi BECs was significantly increased(P < 0.05).4.Transcription factor ESR1 promotes Hi BECs apoptosis and inhibits cell proliferation by activating S100A6 promoter(1)Compared with NC group,the expression level of ESR1 in Hi BECs after GCDC treatment was significantly increased(P < 0.05).(2)The expression level of S100A6 m RNA in Hi BECs was significantly increased after transfected with ESR1 overexpressing plasmid(P < 0.05);the relative expression of S100A6 m RNA in Hi BECs was significantly decreased after transfected with ESR1 Smart silencer(P < 0.05).(3)The expression levels of S100A6,PDC-E2 and ESR1 proteins after ESR1 overexpression in Hi BECs were detected by Western Blotting.Compared with the normal group,the expression levels of S100A6,PDC-E2 and ESR1 proteins in GCDC group were up-regulated(P < 0.05).The protein expression levels of S100A6,PDC-E2 and ESR1 in GCDC + ESR1-OE group with overexpression of ESR1 were significantly higher than those in other groups(P < 0.05).After ESR1 knockdown,S100A6,PDC-E2 and ESR1 protein expression were down-regulated in the GCDC group compared with the normal group(P < 0.05).The protein expression levels of S100A6,PDC-E2 and ESR1 in GCDC + ESR1-KD group were significantly lower than those in other groups(P < 0.05).(4)Dual luciferase reporter gene assay showed that the activity of wild-type p GL3basic-S100A6 was significantly increased in the group with overexpression of ESR1,while the mutant p GL3 basic-S100A6-Luci constructed by point mutation method showed no significant change in the group with overexpression of ESR1.(5)Compared with control + si-control group,the apoptosis percentage of Hi BECs in ESR1 + si-control group was significantly higher than those in control and ESR1 +S100A6-si group(P < 0.05),and cell proliferation was significantly reduced(P < 0.05).5.S100A6 and related lnc RNAs were evaluated as diagnosis and staging markers in patients with PBC(1)Lnc RNAs selecting: Lnc RNAs LINC00312 LINC00472 and LINC01257 related to S100A6 were screened by bioinformatics methods.(2)Relative expression of log10 ESR1 m RNA and S100A6 m RNA was positively correlated(r = 0.367,P = 0.001).(3)Relative expression levels of S100A6 m RNA,Log10 LINC00472 and LINC1257 in plasma of PBC patients were significantly up-regulated compared with healthy controls(P < 0.0001);The mean expression level of LINC00312 was significantly lower than that in healthy controls(P < 0.0001).(4)Expression of S100A6 m RNA in PBC advanced stage(III and IV)was up-regulated compared with in healthy controls(P < 0.0001)and early stage(I and II)(P < 0.0001).The expression level of S100A6 m RNA in the early stage was higher than that in HCs(P= 0.03).Compared with HCS,the expression of LINC01257 was up-regulated in both early and advanced stage(P < 0.0001).The expression level of LINC00312 in advanced PBC was lower than those in early stage and HCs(P = 0.01 and P < 0.0001,respectively).Meanwhile,the expression level of LINC00312 in early stage was lower than that in HCs(P < 0.0001).The expression level of log10 LINC00472 in advanced stage was higher than those in early stage(P < 0.0001)and HCs(P < 0.0001).(5)The AUC of S100A6,LINC00312,log10 LINC00472 and LINC01257 for PBC diagnosis were 0.759,0.7292,0.6942 and 0.7158,respectively.The AUC of the four genes for PBC staging was 0.666,0.661,0.839 and 0.5549,respectively.(6)Relative expression of S100A6 m RNA was positively correlated with log10LINC00472(r = 0.683,P < 0.0001).Serum C-IV level was positively correlated with the relative expression of log10 LINC00472(r = 0.482,P < 0.0001).The relative expression of S100A6 m RNA was positively correlated with C-IV level(r = 0.732,P < 0.0001).(7)Pair t-test was used to analyze and compare the changes in the expression levels of these four genes before and after one year of treatment.The relative expression levels of S100A6 m RNA,log10 LINC00472,LINC01257 were significantly decreased(P values were 0.0018 0.036 and 0.0006,respectively).Meanwhile,the relative expression level of LINC00312 was significantly increased after treatment compared with that before treatment(P = 0.0007).(8)According to the relative expression of the log10 LINC00472 cutoff value(2.33),PBC patients in the L1 subgroup,the relative expression of S100A6 m RNA and serum C-IV level were lower(P < 0.0001).Meanwhile,the relative expression level of LINC01257 in L1 group was higher than that in L2 subgroup(P = 0.005).(9)Independent PBC cohort data were used to construct ROC curves to verify the value of biomarkers in predicting PBC diagnosis and staging.The AUC of S100A6 m RNA,LINC00312,log10 LINC00472 and LINC01257 for PBC diagnosis were 0.769,0.772,0.755 and 0.695,respectively.In addition,the AUC of log10 LINC00472 for PBC staging was 0.835.Conclusion:1.CCL5,IL7 R,TNFRSF1A and immune response pathways as well as molecular simulation may play an important role in the pathogenesis of PBC.These genes and pathways may become new targets for the treatment of PBC.2.The expression of S100A6 was up-regulated in intrahepatic bile duct epithelial cells of a cholestasis mouse model.3.S100A6 promotes the apoptosis of Hi BECs with cholestasis,and inhibits its cell proliferation,which plays an important role in the injury of biliary epithelial cells with cholestasis.4.Transcription factor ESR1 promotes apoptosis and inhibits proliferation of human intrahepatic biliary epithelial cells by activating S100A6 promoter.5.ESR1/S100A6 signaling pathway plays an important role in the damage of PBC cholestasis intrahepatic biliary epithelial cells.6.Plasma S100A6 m RNA,LINC00312,LINC00472 and LINC01257 in PBC patients can be used as potential biomarkers for PBC diagnosis,and LINC00472 can be used as a potential biomarker for PBC staging.