Human Intrahepatic Biliary Epithelial Cells Research Articles

S100 calcium binding protein A6 and associated long noncoding ribonucleic acids as biomarkers in the diagnosis and staging of primary biliary cholangitis.

BACKGROUNDPrimary biliary cholangitis (PBC) is a chronic and slowly progressing cholestatic disease, which causes damage to the small intrahepatic bile duct by immuno-regulation, and may lead to cholestasis, liver fibrosis, cirrhosis and, eventually, liver failure.AIMTo explore the potential diagnosis and staging value of plasma S100 calcium binding protein A6 (S100A6) messenger ribonucleic acid (mRNA), LINC00312, LINC00472, and LINC01257 in primary biliary cholangitis.METHODSA total of 145 PBC patients and 110 healthy controls (HCs) were enrolled. Among them, 80 PBC patients and 60 HCs were used as the training set, and 65 PBC patients and 50 HCs were used as the validation set. The relative expression levels of plasma S100A6 mRNA, long noncoding ribonucleic acids LINC00312, LINC00472 and LINC01257 were analyzed using quantitative reverse transcription-polymerase chain reaction. The bile duct ligation (BDL) mouse model was used to simulate PBC. Then double immunofluorescence was conducted to verify the overexpression of S100A6 protein in intrahepatic bile duct cells of BDL mice. Human intrahepatic biliary epithelial cells were treated with glycochenodeoxycholate to simulate the cholestatic environment of intrahepatic biliary epithelial cells in PBC.RESULTSThe expression of S100A6 protein in intrahepatic bile duct cells was up-regulated in the BDL mouse model compared with sham mice. The relative expression levels of plasma S100A6 mRNA, log10 LINC00472 and LINC01257 were up-regulated while LINC00312 was down-regulated in plasma of PBC patients compared with HCs (3.01 ± 1.04 vs 2.09 ± 0.87, P < 0.0001; 2.46 ± 1.03 vs 1.77 ± 0.84, P < 0.0001; 3.49 ± 1.64 vs 2.37 ± 0.96, P < 0.0001; 1.70 ± 0.33 vs 2.07 ± 0.53, P < 0.0001, respectively). The relative expression levels of S100A6 mRNA, LINC00472 and LINC01257 were up-regulated and LINC00312 was down-regulated in human intrahepatic biliary epithelial cells treated with glycochenodeoxycholate compared with control (2.97 ± 0.43 vs 1.09 ± 0.08, P = 0.0018; 2.70 ± 0.26 vs 1.10 ± 0.10, P = 0.0006; 2.23 ± 0.21 vs 1.10 ± 0.10, P = 0.0011; 1.20 ± 0.04 vs 3.03 ± 0.15, P < 0.0001, respectively). The mean expression of S100A6 in the advanced stage (III and IV) of PBC was up-regulated compared to that in HCs and the early stage (II) (3.38 ± 0.71 vs 2.09 ± 0.87, P < 0.0001; 3.38 ± 0.71 vs 2.57 ± 1.21, P = 0.0003, respectively); and in the early stage (II), it was higher than that in HCs (2.57 ± 1.21 vs 2.09 ± 0.87, P = 0.03). The mean expression of LINC00312 in the advanced stage was lower than that in the early stage and HCs (1.39 ± 0.29 vs 1.56 ± 0.33, P = 0.01; 1.39 ± 0.29 vs 2.07 ± 0.53, P < 0.0001, respectively); in addition, the mean expression of LINC00312 in the early stage was lower than that in HCs (1.56 ± 0.33 vs 2.07 ± 0.53, P < 0.0001). The mean expression of log10 LINC00472 in the advanced stage was higher than those in the early stage and HCs (2.99 ± 0.87 vs 1.81 ± 0.83, P < 0.0001; 2.99 ± 0.87 vs 1.77 ± 0.84, P < 0.0001, respectively). The mean expression of LINC01257 in both the early stage and advanced stage were up-regulated compared with HCs (3.88 ± 1.55 vs 2.37 ± 0.96, P < 0.0001; 3.57 ± 1.79 vs 2.37 ± 0.96, P < 0.0001, respectively). The areas under the curves (AUC) for S100A6, LINC00312, log10 LINC00472 and LINC01257 in PBC diagnosis were 0.759, 0.7292, 0.6942 and 0.7158, respectively. Furthermore, the AUC for these four genes in PBC staging were 0.666, 0.661, 0.839 and 0.5549, respectively. The expression levels of S100A6 mRNA, log10 LINC00472, and LINC01257 in plasma of PBC patients were decreased (2.35 ± 1.02 vs 3.06 ± 1.04, P = 0.0018; 1.99 ± 0.83 vs 2.33 ± 0.96, P = 0.036; 2.84 ± 0.92 vs 3.69 ± 1.54, P = 0.0006), and the expression level of LINC00312 was increased (1.95 ± 0.35 vs 1.73 ± 0.32, P = 0.0007) after treatment compared with before treatment using the paired t-test. Relative expression of S100A6 mRNA was positively correlated with log10 LINC00472 (r = 0.683, P < 0.0001); serum level of collagen type IV was positively correlated with the relative expression of log10 LINC00472 (r = 0.482, P < 0.0001); relative expression of S100A6 mRNA was positively correlated with the serum level of collagen type IV (r = 0.732, P < 0.0001). The AUC for the four biomarkers obtained in the validation set were close to the training set.CONCLUSIONThese four genes may potentially act as novel biomarkers for the diagnosis of PBC. Moreover, LINC00472 acts as a potential biomarker for staging in PBC.

MicroRNA-34a Promotes EMT and Liver Fibrosis in Primary Biliary Cholangitis by Regulating TGF-β1/smad Pathway.

Background and Aims Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease. We found microRNA-34a (miR-34a), as the downstream gene of p53, was overexpressed in some of fibrogenic diseases. In this study, we sought to explore whether miR-34a plays a role in the fibrosis of PBC. Methods The peripheral blood of PBC patients and controls was collected to analyze the level of miR-34a. Human intrahepatic biliary epithelial cells (HIBEC) were cultured. The expression of miR-34a was regulated by miR-34a mimics and inhibitor. The biomarkers of epithelium-mesenchymal transition (EMT), fibrogenesis, inflammation, and transforming growth factor- (TGF-) β1/smad pathway were analyzed. Results We found that miR-34a was overexpressed in the peripheral blood in PBC patients. In vitro, overexpressed miR-34a increased the EMT and fibrogenesis activity of HIBEC. Transforming growth factor-beta type 1 receptor (TβR1), TGF-β1, and p-smad2/3 were upregulated by miR-34a. Inflammatory factors such as IL-6 and IL-17 were also upregulated. Finally, we showed that miR-34a promoted EMT and liver fibrosis in PBC by targeting the TGF-β1/smad pathway antagonist transforming growth factor-beta-induced factor homeobox 2 (TGIF2). Conclusions Our findings show that miR-34a plays an important role in the EMT and fibrosis of PBC through the TGF-β1/smad pathway by targeting TGIF2. This study suggests that miR-34a may be a new marker of fibrogenesis in PBC. Inhibition of miR-34a may be a promising strategy in treating PBC and improving the prognosis of the disease.

Ursodeoxycholic acid impairs liver-infiltrating T-cell chemotaxis through IFN-γ and CX3CL1 production in primary biliary cholangitis.

Ursodeoxycholic acid (UDCA) is the primary treatment for primary biliary cholangitis (PBC), but its mechanism of action remains unclear. Studies suggest that UDCA enhances NF erythroid 2-related factor 2 (NFE2L2) expression and that the interaction between IFN-γ and C-X3-C motif chemokine ligand 1 (CX3CL1) facilitates biliary inflammation in PBC. Therefore, we examined the effects of UDCA on the expression of IFN-γ and CX3CL1 in in vitro and in vivo PBC models such as human liver tissue, a murine model, cell lines, and isolated human intrahepatic biliary epithelial cells (IHBECs). We observed a significant decrease in IFN-γ mRNA levels and positive correlations between IFN-γ and CX3CL1 mRNA levels post-UDCA treatment in PBC livers. NFE2L2-mediated transcriptional activation was significantly enhanced in UDCA-treated Jurkat cells. In 2-octynoic acid-immunized mice, IFN-γ production by liver-infiltrating T cells was dependent on NFE2L2 activation. IFN-γ significantly and dose-dependentlyinduced CX3CL1 expression, which was significantly decreased in HuCC-T1 cells and IHBECs upon UDCA treatment. These results suggest that UDCA-induced suppression of IFN-γ and CX3CL1 production attenuates the chemotactic and adhesive abilities of liver-infiltrating T cells in PBC.

Deletion ofSox9in the liver leads to hepatic cystogenesis in mice by transcriptionally downregulatingSec63

Hepatic cysts are found in heterogeneous disorders with different pathogeneses, of which simple hepatic cysts and polycystic liver diseases are two major types. The process of hepatic cytogenesis for these two diseases is caused by defects in remodelling of the ductal plate during biliary tract development, which is called ductal plate malformation. SOX9 is a transcription factor participating in the process of bile duct development, and thus, its dysregulation may play important roles in hepatic cystogenesis. SEC63 encodes an endoplasmic reticulum membrane protein that is mutated in human autosomal dominant polycystic liver disease. However, the transcriptional regulation of SEC63 is largely unknown. In the present study, a liver-specific Sox9 knockout (Sox9LKO ) mouse was generated to investigate the roles and underlying mechanism of SOX9 in hepatic cystogenesis. We found that hepatic cysts began to be observed in Sox9LKO mice at 6 months of age. The number and size of cysts increased with age in Sox9LKO mice. In addition, the characteristics of hepatic cytogenesis, including the activation of proliferation, absence of primary cilium, and disorder of polarity in biliary epithelial cells, were detected in the livers of Sox9LKO mice. RNAi silencing of SOX9 in human intrahepatic biliary epithelial cells (HIBEpic) resulted in increased proliferation and reduced formation of the primary cilium. Moreover, Sec63 was downregulated in primary biliary epithelial cells from Sox9LKO mice and SEC63 in HIBEpic transfected with siSOX9. Chromatin immunoprecipitation assays and luciferase reporter assays further demonstrated that SOX9 transcriptionally regulated the expression of SEC63 in biliary epithelial cells. Importantly, the overexpression of SEC63 in HIBEpic partially reversed the effects of SOX9 depletion on the formation of primary cilia and cell proliferation. These findings highlight the biological significance of SOX9 in hepatic cytogenesis and elucidate a novel molecular mechanism underlying hepatic cytogenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Long Noncoding RNA GAS5 Accelerates Cholangiocarcinoma Progression by Regulating hsa-miR-1297.

BackgroundLong noncoding RNAs (lncRNAs) have been reported as important molecules in cholangiocarcinoma (CCA) occurrence and development. A previous study showed that lncRNA GAS5 (GAS5) was an oncogene in some tumors. But the role of GAS5 in CCA progression reminds unclear. This research was designed to study the expression and potential effects of GAS5 in the progression of CCA.MethodsThe expression of GAS5 in CCA tissues was evaluated through mining of the TCGA and GEPIA databases. qRT-PCR was applied to validate the results in our clinical samples. χ2 test was used to analyze the association between the expression level of tissue GAS5 and different clinicopathological parameters of CCA patients. The target gene of GAS5 was predicted by bioinformatic databases, and further verified by luciferase reporter assays. Finally, the role of GAS5 in CCA cells invasion and proliferation was detected by Transwell assay and CCK-8 assay.ResultsCompared to the adjacent nontumor tissues and the normal human intrahepatic biliary epithelial cell, the expression of GAS5 was markedly increased in CCA tissues (p<0.001) and cell lines (p<0.01), respectively. CCA patients with high GAS5 expression tended to present lymph node metastasis (p<0.001) and had advanced clinical stage (p=0.006). The bioinformatics analysis predicted that hsa-miR-1297 was the potential target gene of GAS5, which was validated by luciferase reporter assays. In addition, the function study showed that GAS5 acted as a “sponge” to downregulate hsa-miR-1297, thus modulating CCA cell proliferation and invasion.ConclusionGAS5 acts as an endogenous sponge of hsa-miR-1297 to promote CCA cell proliferation and invasion, which might be a potential biomarker and therapeutic target for CCA.

MC-LR-induced interaction between M2 macrophage and biliary epithelial cell promotes biliary epithelial cell proliferation and migration through regulating STAT3.

Microcystin-leucine-arginine (MC-LR) was produced by toxic cyanobacteria, which has been shown to have potent hepatotoxicity. Our previous study has proved that MC-LR were able to promote intrahepatic biliary epithelial cell excessive proliferation. However, the underlying mechanism is not yet entirely clarified. Herein, mice were fed with different concentrations (1, 7.5, 15, or 30μg/L) of MC-LR by drinking water for 6months. As the concentration of MC-LR increased, a growing number of macrophages were evaluated in the portal area of the mouse liver. Next, we built a co-culture system to explore the interaction between macrophages (THP-1 cells) and human intrahepatic biliary epithelial cells (HiBECs) in the presence of MC-LR. Under the exposure of MC-LR, HiBECs secreted a large amount of inflammatory factors (IL-6, IL-8, IL-1β, COX-2, XCL-1) and chemokine (MCP-1), which produced a huge chemotactic effect on THP-1 cells and induced elevation of the surface M2-subtype biomarkers (IL-10, CD163, CCL22, and Arg-1). In turn, high content of IL-6 in the medium activated JAK2/STAT3, MEK/ERK, and PI3K/AKT pathways in HiBECs, inducing HiBEC abnormal proliferation and migration. Together, these results suggested that MC-LR-mediated interaction between HiBECs and macrophages induced the M2-type polarization of macrophages, and activated IL-6/JAK2/STAT3, MEK/ERK, and PI3K/AKT pathways in HiBECs, further enhanced cell proliferation, improved cell migration, and hindered cell apoptosis by activating p-STAT3. MC-LR stimulates HiBECs to produce various inflammatory factors, recruiting a large number of macrophages and promoting the differentiation of macrophages into M2-type. In turn, the M2 macrophages could also produce amounts of IL-6 and activate STAT3 through JAK2/STAT3, MEK/ERK, and PI3K/AKT pathways in HiBECs, resulting in the promotion of cell proliferation, inhibition of apoptosis, and enhancement of migration.

FGF1 receptor antagonist decreases biliary proliferation, fibrosis, and senescence in a mouse model of chronic cholestasis

BackgroundAcute or chronic cholestasis is a hallmark for a wide variety of liver diseases such as primary sclerosing cholangitis (PSC), alcoholic liver disease (ALD) and cholestasis of pregnancy. Available treatments for cholestasis are few and the subsequent scarring often necessitates the patients undergo a costly and risky liver transplantion. Fibroblastic growth factor 1 (FGF1) is involved in a wide range of cellular activities and has been previously shown to act in both a protective and detrimental manner as it participates in the fibrotic process of various tissue systems. Therefore, we aimed to evaluate the role of FGF1 in the progression of senescence, inflammation and fibrosis in an MDR−/− mouse model of PSC.MethodsFVB and MDR−/− mice were treated with 100ng/kg/day for 7‐days with the FGF receptor inhibitor, AZD4547. Liver, serum, and pure cholangiocytes were obtained. Hepatic fibrosis was evaluated by H&amp;E and Sirius Red staining, and qPCR for collagen‐1a. The inflammatory response was evaluated by qPCR in pure cells for IL1‐b, IL‐6, and IL‐33 and immunofluorescent staining of F4/80. Biliary proliferation was determined through IHC CK‐19 expression and qPCR for Ki67 and PCNA. Senescence was evaluated by qPCR for p53, p18 and p21. FGFR expression was determined by qPCR. FGF1 serum expression was measured using a commercially available ELISA kit and in OCT liver sections by immunofluorescence (IF). In vitro, human intrahepatic biliary epithelial cells (HIBepiC) were treated with 150ng/ml of either FGF1 or AZD4547 with or without the presence of 100ng/ml of LPS for 24 hr before evaluating inflammatory markers, biliary proliferation and markers of fibrosis.ResultsAs expected, MDR−/− mice had a significant increase in biliary proliferation and fibrosis when compared to the FVB control. MDR−/− animals treated with AZD4547 experienced a significant reduction in fibrosis, with a subsequent decrease in collagen 1a1 gene expression. Treatment with AZD4547 also decreased senescence and inflammatory markers in MDR−/− mice evaluated by IHC and IF. Serum analysis showed a dramatic increase in FGF1 levels that were significantly reduced upon treatment. In vitro studies also had significant reductions in inflammatory and fibrotic markers.ConclusionFGF1 receptor antagonist decreased biliary proliferation, senescence, inflammation and fibrosis in cholangiocytes and surrounding liver in a mouse model of PSC. Targeting the FGFR pathway may provide a therapeutic option for patients suffering from various forms of chronic cholestasis such as PSC.Support or Funding InformationNIH RO1 DK110035R01 DK115184

Sirt6 opposes glycochenodeoxycholate-induced apoptosis of biliary epithelial cells through the AMPK/PGC-1\u03b1 pathway

BackgroundInduction of biliary epithelial cell apoptosis by toxic bile acids is involved in the development of cholestatic disease, but the underlying molecular mechanism is not clear. The purpose of this study was to investigate the molecular mechanisms involved in Sirt6 protection against the apoptosis of human intrahepatic biliary epithelial cells (HiBEC) induced by the bile acid glycochenodeoxycholate (GCDC).ResultsSirt6 was either overexpressed or knocked down in HiBEC, with or without GCDC pretreatment. The CCK-8 assay was used to assess cell viability and, Hoechst 33258 staining was used to determine apoptotic rate. Mitochondrial DNA (mtDNA) copy number, malondialdehyde (MDA) and reactive oxygen species (ROS) production were detected to evaluate the severity of the mitochondrial dysfunction and oxidative stress. The mRNA and protein levels of PGC-1α, Nrf1, and Nrf2 were analyzed using RT-qPCR and western blot assay. The results showed that Sirt6 opposed GCDC-induced apoptosis in HiBEC via up-regulating PGC-1α expression and stabilizing mtDNA. We used agonists and inhibitors of AMPK to demonstrate that Sirt6 increased PGC-1α expression through the AMPK pathway whereas GCDC had the opposite effect. Finally, western blot, luciferase assay, and co-immunoprecipitation were used to describe a direct interaction and acetylation modification of PGC-1α by Sirt6.ConclusionOur data illuminated that Sirt6 ameliorated GCDC-induced HiBEC apoptosis by upregulating PGC-1α expression through the AMPK pathway and its deacetylation effect.

Long non-coding RNA myocardial infarction associated transcript promotes the proliferation of cholangiocarcinoma cells by targeting miR-551b-3p/CCND1 axis.

Accumulating reports have demonstrated that long non-coding RNAs (lncRNAs) play critical roles in the occurrence and metastasis of cholangiocarcinoma (CCA). LncRNA myocardial infarction associated transcript (MIAT) has been widely reported in hepatocellular carcinoma, pancreatic cancer and colorectal cancer, but the relationship between MIAT and CCA progression has not yet been investigated. In the present study, we found that the expression of MIAT in CCA tissues was prominently higher than that in normal bile duct tissues. Moreover, TCGA-CHOL data in the GEPIA platform further revealed the upregulated expression of MIAT in CCA tissues. Additionally, quantitative real-time PCR results showed that MIAT expression was increased in CCA cell lines compared to the human intrahepatic biliary epithelial cell line. Functionally, MIAT knockdown significantly inhibited cell proliferation and induced G0/G1 phase arrest as well as apoptosis in HuCCT-1 and QBC939 cells. Conversely, ectopic expression of MIAT obviously facilitated the proliferation, cell cycle progression and apoptosis resistance of RBE cells. Mechanistically, MIAT directly interacted with miR-551b-3p and inversely modulated miR-551-3p level in CCA cells. Furthermore, MIAT knockdown reduced the expression of cyclin D1 (CCND1), which was rescued by miR-551b-3p silencing in HuCCT-1 cells. Importantly, CCND1 restoration partially reversed MIAT knockdown-induced proliferation inhibition, G0/G1 phase arrest and apoptosis in HuCCT-1 cells. In conclusion, MIAT was frequently overexpressed in CCA. MIAT contributed to the growth of CCA cells by targeting miR-551b-3p/CCND1 axis.

Overexpression of microRNA-96 is associated with poor prognosis and promotes proliferation, migration and invasion in cholangiocarcinoma cells via MTSS1.

MicroRNA-96 (miR-96) has been revealed serve an oncogenic role in various types of cancer. However, the role of miR-96 in cholangiocarcinoma (CCA) development and progression is yet to be elucidated. Thus, the aim of the present study was to investigate the role of miR-96 in CCA. The expression pattern of miR-96 in CCA tissues and cell lines was evaluated using reverse transcription-quantitative PCR analysis. Kaplan-Meier curves and Cox regression analyses were conducted to investigate the prognostic significance of miR-96 in CCA. Cell Counting Kit-8 and Transwell assays were performed to identify the functions of miR-96. The association between miR-96 and metastasis suppressor-1 (MTSS1) was verified using a dual-luciferase assay. The results demonstrated that miR-96 expression levels were increased in CCA tissues and cell lines compared with those in adjacent normal tissues and normal human intrahepatic biliary epithelial cell lines, respectively. High expression levels of miR-96 were significantly associated with lymph node metastasis, differentiation and TNM stage. In addition, upregulated expression of miR-96 was associated with a poorer prognosis and was predicted to be a prognostic factor in patients with CCA. Overexpression of miR-96 in vitro promoted CCA cell proliferation, migration and invasion. Additionally, MTSS1 was identified as a direct target of miR-96. The results of the present study indicated the clinical and biological importance of miR-96 as an oncogene in CCA. miR-96 may represent an independent prognostic biomarker and may promote CCA cell proliferation, migration and invasion by targeting MTSS1.

Abnormal Expression of ERα in Cholangiocytes of Patients With Primary Biliary Cholangitis Mediated Intrahepatic Bile Duct Inflammation.

ERα, one of the classical receptors of estrogen, has been found to be abnormally up-regulated in patients with primary biliary cholangitis (PBC), which is an important factor leading to ductopenia. ERα-mediated signaling pathways are involved in proliferation of human intrahepatic biliary epithelial cells (HiBECs) and portal inflammation. Our previous studies have shown that the expression levels of ERα in the liver tissues of PBC patients are positively correlated with the levels of serum pro-inflammatory cytokines. The present study was designed to assess the relationship between abnormal ERα expression in small bile ducts and the progression of PBC. We examined the levels of multiple cytokines and analyzed their relationship with clinical parameters of livers functions in a cohort of 43 PBC patients and 45 healthy controls (HC). The levels of ERα expression and the relation with the levels of cytokines were further assessed. The localization of cytokines and ERα-mediated signaling pathways in liver were examined using immunohistochemistry. The possible underlying mechanisms of these alterations in PBC were explored in vitro. Our results demonstrated that the levels of IL-6, IL-8, and TNF-α were increased in PBC patients, and positively correlated with the serum AKP levels and ERα expression levels. Moreover, the expression of these cytokines were up-regulated in HiBECs that were stimulated with 17β-estradiol and PPT (an ERα agonist) and they also were positive in intrahepatic bile duct of PBC patients. The ERα-mediated expression of pro-inflammatory cytokines was induced by JNK, P38, and STAT3 phosphorylation in HiBECs. In addition, the CD54 expression was increased in HiBECs after ERα activation, which induced peripheral blood monouclear cells (PBMCs) recruitment. In conclusion, the present study highlighted a key role of abnormal ERα expression in inducing an inflammatory phenotype of HiBECs, which was critical in the development of inflammation and damage in small bile duct.

Identification of TPD52 and DNAJB1 as two novel bile biomarkers for cholangiocarcinoma by iTRAQ‑based quantitative proteomics analysis.

Cholangiocarcinoma (CCA) represents a type of epithelial cancer with a late diagnosis and poor outcome. However, the molecular mechanisms responsible for the development of CCA have not yet been fully identified. Thus, in this study, we aimed to elucidate some of these mechanisms. For this purpose, isobaric tags for relative and absolute quantification (iTRAQ) was performed to analyze the secretory proteins from the 2 CCA cell lines, TFK1 and HuCCT1, as well as from a normal biliary epithelial cell line, human intrahepatic biliary epithelial cells (HiBECs). Differentially expressed proteins (DEPs) were identified and biological process analysis was performed according to the Gene Ontology (GO) functional classification annotation and KEGG metabolic pathway map analysis. tumor protein D52 (TPD52) and DnaJ heat shock protein family (Hsp40) member B1 (DNAJB1) were validated using RT-qPCR, western blot analysis and immunohistochemistry. In total, 778 proteins were identified as DEPs. Following validation, TPD52 and DNAJB1 were used for further analysis. The expression levels of TPD52 and DNAJB1 were elevated in CCA cell lines, tissues and bile samples, suggesting that these proteins may contribute to tumor pathogenesis. In addition, the expression levels of TPD52 and DNAJB1 were found to be closely associated with the clinical parameters and prognosis of patients with CCA. On the whole, the findings of this study indicate that TPD52 and DNAJB1 may serve as novel bile biomarkers for CCA.

Knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes in the Mdr2−/− mouse model of primary sclerosing cholangitis (PSC)

BackgroundCholangiocytes are the target cells of cholangiopathies including primary sclerosing cholangitis (PSC). Vimentin is an intermediate filament protein that has been found in various types of mesenchymal cells. The aim of this study is to evaluate the role of vimentin in the progression of biliary damage/liver fibrosis and whether there is a mesenchymal phenotype of cholangiocytes in the Mdr2−/− model of PSC.MethodsIn vivo studies were performed in 12 wk. Mdr2−/− male mice with or without vimentin Vivo-Morpholino treatment and their corresponding control groups. Liver specimens from human PSC patients, human intrahepatic biliary epithelial cells (HIBEpiC) and human hepatic stellate cell lines (HHSteCs) were used to measure changes in epithelial-to-mesenchymal transition (EMT).FindingsThere was increased mesenchymal phenotype of cholangiocytes in Mdr2−/− mice, which was reduced by treatment of vimentin Vivo-Morpholino. Concomitant with reduced vimentin expression, there was decreased liver damage, ductular reaction, biliary senescence, liver fibrosis and TGF-β1 secretion in Mdr2−/− mice treated with vimentin Vivo-Morpholino. Human PSC patients and derived cell lines had increased expression of vimentin and other mesenchymal markers compared to healthy controls and HIBEpiC, respectively. In vitro silencing of vimentin in HIBEpiC suppressed TGF-β1-induced EMT and fibrotic reaction. HHSteCs had decreased fibrotic reaction and increased cellular senescence after stimulation with cholangiocyte supernatant with reduced vimentin levels.InterpretationOur study demonstrated that knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes, which leads to decreased biliary senescence and liver fibrosis. Inhibition of vimentin may be a key therapeutic target in the treatment of cholangiopathies including PSC.FundNational Institutes of Health (NIH) awards, VA Merit awards.

RANK/RANKL Acts as a Protective Factor by Targeting Cholangiocytes in Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by the highly selective autoimmune injury of small intrahepatic bile ducts. Studies reported that the cholangiocytes from PBC patients expressed significantly higher levels of both receptor activator of nuclear factor-kappa B (RANK) and its ligand RANKL. However, the accurate role of RANK/RANKL axis in PBC remains unclear. Forty patients with PBC were enrolled according to the inclusion criteria. The biochemical parameters (alkaline phosphatase, ALP; gamma-glutamyltransferase, GGT; alanine aminotransferase, ALT; aspartate transaminase, AST; total bilirubin, TB) were collected at baseline and followed-up after 6months of treatment with ursodeoxycholic acid (UDCA, 15mg/kgd). Stages of PBC were diagnosed based on liver biopsy histopathology according to Nakanuma's criteria. RANK expression in hepatic tissues was detected by immunohistochemistry. The cellular immunofluorescence method was used to locate the distribution of RANK in the human intrahepatic biliary epithelial cells (HIBECs) cultured in vitro. HIBECs were treated with RANKL at a concentration of 100ng/ml or transfected with RANK-overexpressing lentivirus (LV-RANK). CCK-8 assay and cell cycle assay were used to detect the cell proliferation. Real-time PCR was used to detect the expression of IL-6, E-cadherin, VCAM, ICAM-1, TNF-α, and CD80. RANK expression in liver biopsies from early PBC patients (stage I + stage II) was significantly lower than that from advanced PBC patients (stage III + stage IV) (1.7 ± 0.63 vs. 2.3 ± 0.45 scores, P < 0.05). High-RANK patients seemed to have better response to UDCA than low-RANK patients (88.9% vs. 40.9%, P < 0.05). The baseline biochemical parameters between the two groups were comparable. The decline percentages of ALP and GGT after UDCA treatment were more obvious in high-RANK patients than those in low-RANK patients (53.90% ± 9.82% vs. 23.93% ± 6.24%, P < 0.05; 74.11% ± 7.18% vs. 48.00% ± 8.17%, P < 0.05, respectively). HIBECs proliferation was significantly inhibited after treatment of RANKL or transfection with LV-RANK. Increased expression of IL-6 and E-cadherin was observed in HIBECs treated with RANKL or LV-RANK. The overall hepatic RANK expression was associated with disease severity and biochemical response in PBC patients. Activation of RANK/RANKL signaling pathway inhibited cholangiocytes proliferation in vitro. Our study suggested that RANK/RANKL pathway might be a potential target of immunotherapy of PBC based on its involvement in the occurrence and development of the disease.

Long non-coding RNA UCA1 promotes proliferation and invasion of intrahepatic cholangiocarcinoma cells through targeting microRNA-122.

Long non-coding RNA urothelial carcinoma-associated 1 (UCA1) has a role in various common types of human malignancy, including cholangiocarcinoma; however, the expression and function of UCA1 in intrahepatic cholangiocarcinoma (ICC) has remained elusive. In the present study, it was observed that UCA1 expression was significantly upregulated in ICC tissues and cell lines compared with that in the adjacent non-tumour tissues and a human intrahepatic biliary epithelial cell line, respectively. The increased expression of UCA1 was significantly associated with lymph node metastasis and clinical T-stage in ICC. Furthermore, the ICC patients with high expression of UCA1 had a shorter survival time when compared with that of patients with low UCA1 expression. Knockdown of UCA1 caused a significant decrease in ICC cell proliferation and invasion, while ectopic overexpression of UCA1 significantly promoted the proliferation and invasion of ICC cells. Furthermore, it was revealed that UCA1 directly binds to microRNA (miR)-122 to negatively regulate its expression in ICC cells. In addition, miR-122 mimics abrogated the promoting effects of UCA1 on ICC cell proliferation and invasion. In addition, an inverse correlation between miR-122 and UCA1 expression in ICC tissues was observed. In conclusion, the present study demonstrates that the lncRNA UCA1 promotes ICC cell proliferation and invasion at least in part by targeting miR-122, suggesting that the UCA1/miR-122 interaction may become a potential therapeutic target for the treatment of ICC.

IL-37 suppresses migration and invasion of gallbladder cancer cells through inhibition of HIF-1α induced epithelial-mesenchymal transition.

Gallbladder carcinoma (GBC) is the seventh most common cancer across the globe and the most common malignancy of the biliary tract. Epithelial-mesenchymal transition (EMT) is an important pre-requisite for tumor metastasis; however, its mechanism in GBC has not yet been defined. In the present study, we investigated the effects of interleukin-37 (IL-37) on the epithelial-mesenchymal transition (EMT) of gallbladder cancer cells. RT-qPCR and Western blotting were used to determine the expression of IL-37 in GBC cancer cells and non-tumorigenic human intra-hepatic biliary epithelial cell line. Western blotting was also used for detecting the expression of vimentin, Snail, and E-cadherin. Expression level of IL-37 in GBC cells was decreased in GBC cancer cells compared with the non-tumorigenic human intra-hepatic biliary epithelial cell line. Decreased expression of vimentin and Snail and increased expression of E-cadherin were found in the groups which overexpress IL-37 when compared with the control. Mechanism study showed that IL-37 suppressed the expression of HIF1α in cells. However, HIF1α stabilization by CoCl2 could attenuate the function of IL-37. Our results indicate that IL-37 plays an antitumor role during the progression of gallbladder carcinoma. IL-37 could inhibit HIF1α induced EMT. Our data provide a new strategy for the treatment of gallbladder cancer.

Long non-coding RNA HOTAIR promotes tumorigenesis and forecasts a poor prognosis in cholangiocarcinoma

Cholangiocarcinoma (CCA) arising from the neoplastic transformation of cholangiocytes with increasing incidence in the worldwide. Unfortunately, a large amount of CCA patients lost their chance for surgery because it is hard to diagnose in the early stages. Long non-coding RNAs (lncRNAs) is closely associated with development and progression of various malignant tumors. Hox transcript antisense intergenic (HOTAIR), a negative prognostic factor for patients with gastric, liver and pancreatic carcinoma. Its transcription levels and functional roles in CCA is still unknown. Therefore, we aimed to explore the effect of HOTAIR in CCA including cell proliferation, apoptosis, migration, invasion and epithelial-to-mesenchymal transition (EMT). The results showed that HOTAIR was highly expressed both in CCA tissue samples and cell lines compared with corresponding normal bile duct tissues and Human intrahepatic biliary epithelial cells (HIBEC). Its overexpression was closely correlated with Tumor size, TNM stage and postoperative recurrence in CCA patients. Moreover, up-regulation of HOTAIR has correlation with prognosis in CCA patients. Knockdown of HOTAIR by siRNAs significantly decreased the migration and invasion but increased apoptosis of CCA cells in vitro. Overall, our study revealed that HOTAIR may play as a new potential therapeutic target and forecast poor prognosis for this fatal disease.

Mesenchymal stem cells enhance autophagy of human intrahepatic biliary epithelial cells in vitro.

The present findings constitute the first report that human umbilical cord-derived MSCs enhance autophagic flux in HiBECs through a STAT3-dependent way: MSCs enhance the autophagic flux by increasing the formation of autophagosome and autolysosome in GCDC-treated HiBECs. MSCs decrease the STAT3 activity and the expression of eIF2α in GCDC-treated HiBECs; in addition, MSCs increase the expression of PKR. With STAT3 silencing, MSCs enhance neither the levels of LC3II nor the expression of PKR in GCDC-treated HiBECs.

Role of cantharidin in the activation of IKKα/IκBα/NF‑κB pathway by inhibiting PP2A activity in cholangiocarcinoma cell lines.

Cantharidin (CAN), a potent inhibitor of serine/threonine-protein phosphatase 2A (PP2A), is widely used in clinical practice, particularly in the treatment of advanced cancer due to its specific action on these types of cancer. In the present study, the inhibitory effect of CAN was examined in two cholangiocarcinoma cell lines (QBC939 and Hucc-t1). Following treatment with CAN, cell viability was effectively reduced in QBC939 and Hucc-t1 cells and normal human intrahepatic biliary epithelial cells. However, a slight increase in reactive oxygen species levels in QBC939 cells treated with CAN was observed post-treatment. CAN significantly inhibited cell migration and invasion in a dose-dependent manner. Western blot analysis demonstrated that the nuclear factor-κB (NF-κB) pathway was stimulated by CAN, which was confirmed by the upregulated phosphorylation levels of inhibitor of NF-κB kinase subunit α (IKKα) and NF-κB inhibitor α (IκBα) in cells, and an increased NF-κB p65 subunit level in the nucleus. The expression levels of 72 kDa type IV collagenase (MMP2) and matrix metalloproteinase 9 (MMP9) were downregulated by CAN. Notably, there was a negative association between MMP2 and MMP9 expression levels, and NF-κB p65, although NF-κB p65 regulates the expression of MMP2 and MMP9 and has a positive association with these proteins in various types of cancer. Notably, it was observed that CAN exerted specific inhibition on PP2A activity and thereby resulted in the activation of the IKKα/IκBα/NF-κB pathway. Therefore, CAN-induced cell inhibition maybe partially dependent on the activation of the IKKα/IκBα/NF-κB pathway. In conclusion, it was demonstrated that CAN selectively and effectively inhibited cholangiocarcinoma cell migration and invasion. The present study may provide a novel insight into the use of CAN as a therapeutic candidate in the treatment of cholangiocarcinoma.

Methylation-Mediated Silencing of GATA5 Gene Suppresses Cholangiocarcinoma Cell Proliferation and Metastasis

Cholangiocarcinoma (CCA) is one of the most common hepatic and biliary malignancies, accounting for about 3% of all gastrointestinal tumors. GATA5 is a transcription factor capable of suppressing the development of various human cancer types. Transcriptional inactivation and CpG island (CGI) methylation of GATA3 and GATA5, two members of the GATA family of transcription factors, have been observed in some human cancers. But whether high-density CGI methylation of GATA5 is associated with the clinical course of CCA patients has not been clarified. Herein, we observed reduced expression of GATA5 in CCA tissues compared with noncancerous tissues. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored GATA5 expression in CCA cell lines. Furthermore, GATA5 expression was downregulated after treatment with IL-6 in human intrahepatic biliary epithelial cells. Upregulated GATA5 inhibited CCA cell growth and metastasis. Mechanistically, GATA5 suppressed CCA cell growth and metastasis via Wnt/β-catenin pathway. Specific β-catenin inhibitor or siRNA abolished the discrepancy of the proliferation and metastasis capacity between GATA5-overexpression CCA cells and their control cells, which further confirmed that Wnt/β-catenin was required in GATA5-inhibited CCA cell growth and metastasis.