Normal Biliary Epithelial Cells Research Articles

Identification of Novel Cuproptosis-Related Genes Mediating the Prognosis and Immune Microenvironment in Cholangiocarcinoma.

Cuproptosis is a novel type of mediated cell death strongly associated with the progression of several cancers and has been implicated as a potential therapeutic target. However, the role of cuproptosis in cholangiocarcinoma for prognostic prediction, subgroup classification, and therapeutic strategies remains largely unknown. A systematic analysis was conducted among 146 cuproptosis-related genes and clinical information based on independent mRNA and protein datasets to elucidate the potential mechanisms and prognostic prediction value of cuproptosis-related genes. A 10-cuproptosis-related gene prediction model was constructed, and its effects on cholangiocarcinoma prognosis were significantly connected to poor patient survival. Additionally, the expression patterns of our model included genes that were validated with several cholangiocarcinoma cancer cell lines and a normal biliary epithelial cell line. First, a 10-cuproptosis-related gene signature (ADAM9, ADAM17, ALB, AQP1, CDK1, MT2A, PAM, SOD3, STEAP3, and TMPRSS6) displayed excellent predictive performance for the overall survival of cholangiocarcinoma. The low-cuproptosis group had a significantly better prognosis than the high-cuproptosis group with transcriptome and protein cohorts. Second, compared with the high-risk and low-risk groups, the 2 groups displayed distinct tumor microenvironments, reduced proportions of endothelial cells, and increased levels of cancer-associated fibroblasts based on CIBERSORTx and EPIC analyses. Third, patients' sensitivities to chemotherapeutic drugs and immune checkpoints revealed distinctive differences between the 2 groups. Finally, in replicating the expression patterns of the 10 genes, these results were validated with quantitative real-time polymerase chain reaction results validating the abnormal expression pattern of the target genes in cholangiocarcinoma. Collectively, we established and verified an effective prognostic model that could separate cholangiocarcinoma patients into 2 heterogeneous cuproptosis subtypes based on the molecular or protein characteristics of 10 cuproptosis-related genes. These findings may provide potential benefits for unveiling molecular characteristics and defining subgroups could improve the early diagnosis and individualized treatment of cholangiocarcinoma patients.

The Mechanism of Long Non-Coding RNA SNHG7 in Cholangiocarcinoma Cell Proliferation, Migration, and Epithelial-Mesenchymal Transition

Objective: To investigate the mechanism of long non-coding RNA SNHG7 and its regulatory effect on the proliferation, migration, and epithelial-mesenchymal transition of cholangiocarcinoma cells. Methods: A total of 20 pairs of cholangiocarcinoma and adjacent non-tumor bile duct tissues were collected from patients with cholangiocarcinoma who underwent surgery in the Affiliated Hospital of Hebei University (Hebei, China). Cholangiocarcinoma cell lines CCLP-1, QBC939, RBE, and HCC-9810 as well as normal human biliary epithelial cell line HIBEC were purchased for cell culture. We performed cell transfection, quantitative real-time polymerase chain reaction (qRT-PCR) to detect gene expression, Cell Counting Kit-8 (CCK-8) experiment to determine cell proliferation ability, scratch test to determine cell migration ability, and Transwell test to detect cell invasion ability. Results: The expression of lncRNA SNHG7 in cholangiocarcinoma cell lines CCLP-1, QBC939, RBE, and HCCC-9810 was 3.21 ± 1.01, 3.03 ± 1.02, 2.98 ± 1.21, and 3.12 ± 1.14, respectively, while its expression in normal cell line HIBEC was 3.21 ± 1.21; the expression of lncRNA SNHG7 in CCLP-1 was the highest; compared with HIBEC, the p values were all less than 0.05, indicating that the difference was statistically significant. The expression of miR-520f-3p in CCLP-1, QBC939, RBE, and HCCC-9810 was 1.45 ± 0.75, 1.55 ± 0.71, 1.54 ± 0.73, and 1.61 ± 0.72, respectively, while its expression in normal cell line HIBEC was 3.21 ± 1.21; the expression of miR-520f-3p in CCLP-1 was the lowest, and compared with HIBEC, the p values were all less than 0.05, indicating that the difference was statistically significant. In qRT-PCR, the expression of lncRNA SNHG7 of si-NC (3.21±1.11) was significantly higher than that of si-SNHG7 (1.14±0.76), and the p value was less than 0.05, indicating that the difference was statistically significant. In the CCK-8 experiment, the proliferation ability of CCLP-1 cells of the si-NC group at 24 h, 48 h, and 72 h was 0.61±0.59, 0.75±0.68, and 1.36±0.71, respectively; the proliferation ability of CCLP-1 cells of the si-SNHG7 group at 24 h, 48 h, and 72 h was 0.51±0.64, 0.59±0.59, and 0.63±0.61, respectively; there was a significant decrease in the proliferation ability, and the p value was less than 0.05, indicating a statistically significant difference. After 24 h of scratch treatment, compared with the si-NC group, the migration ability of CCLP-1 cells of the si-SNHG20 group was reduced (t = 6.356, P = 0.026). The results of Transwell test showed that the cell invasion ability of CCLP-1 in the si-SNHG20 group was significantly reduced compared with the si-NC group (t = 7.845, P = 0.032). Conclusion: Exploring the gene expression mechanism in relation to the occurrence and development of cholangiocarcinoma is beneficial to future clinical work in terms of diagnosis, treatment, and prognosis. The knockdown of lncRNA SNHG7 can effectively inhibit the proliferation, migration, and invasion of cholangiocarcinoma.

Novel Drug Candidate Prediction for Intrahepatic Cholangiocarcinoma via Hub Gene Network Analysis and Connectivity Mapping

Simple SummaryOnly about 15% of intrahepatic cholangiocarcinoma (ICC) patients meet the criteria for resection at the time of diagnosis. For patients with advanced and/or metastatic disease, the development of novel therapeutic strategies is urgently needed. The aim of our study was to identify possible novel therapeutic targets and drugs for ICC by using transcriptomic profiles from the Gene Expression Omnibus databases and The Cancer Genome Atlas. The weighted co-expression gene network was constructed to screen hub genes. Potential drug candidates with promise in the treatment of ICC were identified by analyzing key protein–protein interaction (PPI) networks of the hub genes to identify potential interacting drugs based on the Connectivity Map database.Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy, and there is a need for effective systemic therapies. Gene expression profile-based analyses may allow for efficient screening of potential drug candidates to serve as novel therapeutics for patients with ICC. The RNA expression profile of ICC and normal biliary epithelial cells were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Function annotation and enrichment pathway analyses of the differentially expressed genes (DEGs) were finished using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A weighted gene co-expression network (WGCN) was constructed by WGCN analysis (WGCNA). Key genes from the DEGs and co-expression gene modules were analyzed to generate a protein–protein interaction (PPI) network. The association between the top 10 screened hub genes and the overall and disease-free survival of ICC patients was examined. The Connectivity Map (cMap) analysis was performed to identify possible drugs for ICC using hub genes. A total of 151 key genes were selected from the overlapping genes of 1287 GSE-DEGs, 8183 TCGA-DEGs and 1226 genes in the mixed modules. A total of 10 hub genes of interest (CTNNB1, SPP1, COL1A2, COL3A1, SMAD3, SRC, VCAN, PKLR, GART, MRPS5) were found analyzing protein–protein interaction. Using the cMap, candidate drugs screened with potential efficacy for ICC included three tyrosine kinase inhibitors (dasatinib, NVP-BHG712, tivantinib), two cannabinoid receptor agonists (palmitoylethanolamide, arachidonamide), two antibiotics (moxifloxacin, amoxicillin), one estrogen receptor agonist (levonorgestrel), one serine/threonine protein kinase inhibitor (MK-2206) and other small molecules. Key genes from network and PPI analysis allowed us to identify potential drugs for ICC. The identification of novel gene expression profiles and related drug screening may accelerate the identification of potential novel drug therapies for ICC.

Abstract 6175: Developing antibody-drug conjugates against cholangiocarcinoma using a novel molecular target

Abstract As a rare malignancy, cholangiocarcinoma (CCA) is a lethal disease with a 5-year overall survival rate of merely 5%. Highly invasive and asymptomatic characteristics of CCA cause most patients diagnosed at advanced stages, severely deteriorating their clinical outcomes. The drug treatment options for CCA are limited and surgical resection of CCA frequently suffers from a high recurrence rate of 66%. Albeit Pemigatinib, a FGFR2 inhibitor, has recently been approved by FDA as the first CCA-targeted therapeutic, only less than 10% of patients with CCA carry FGFR2 fusion mutations that can benefit from this drug. Ivosidenib, another targeted drug against IDH1 mutation, only works for 13% CCA patients carrying this mutation. Therefore, discovering new molecular targets and developing associated targeted drugs remain a significant and unmet medical need in CCA therapy. In this study, we first screened potential molecular targets for CCA by comparing the expression levels of a panel of cancer-related cell membrane antigens between human CCA cells and normal human intrahepatic biliary epithelial cells through flow cytometry. CD54 was identified as a remarkable molecular target of CCA with strong binding force to its antibodies and high endocytosis efficiency by flow cytometry and confocal fluorescent microscopy. Meanwhile, we confirmed that CD54 was absent in most normal human tissues but was highly upregulated in CCA tumor tissues through immunohistochemical (IHC) staining. Based on this newly discovered CCA target, we constructed a series of CD54 antibody-drug conjugates (ADCs) using different chemical linkers and payloads. We next evaluated and compared the half maximum inhibitory concentrations (IC50s) of each ADC in ablating human CCA cells in vitro in order to identify an optimized ADC formulation. Furthermore, we validated its anti-tumor efficacy against primary and peritoneal metastatic CCA tumors in vivo in comparison with conventional chemotherapy. Eventually, we determined the biodistribution and safety profile of this CD54 ADC in order to ensure that it is well-tolerated under the effective dose. In general, we identified CD54 as a novel molecular target for CCA through antibody library screening, and explored its potential in the development of CCA-targeted ADCs, providing a promising targeted therapeutic for clinical treatment of CCA. Citation Format: Bing Zhu, Shi-Li Yao, Xin-Yan Wang, Rui Xu, Peng Guo, Jie-Er Ying. Developing antibody-drug conjugates against cholangiocarcinoma using a novel molecular target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6175.

Differential Expression of Long Non-coding RNA Cancer Susceptibility Candidate 2 and Imprinted Gene H19 in Extrahepatic Cholangiocarcinoma

Objective To investigate the expression and the potential roles of long non-coding RNA(lncRNA)cancer susceptibility candidate 2(CASC2)and imprinted gene H19 in extrahepatic cholangiocarcinoma(ECC). Methods Four samples from patients with ECC were collected for high-throughput sequencing which was conducted to reveal the transcriptomic profiles of lncRNA CASC2 and H19.Bioinformatics tools were employed to predict the potential roles of the two genes.Another 22 ECC tissue samples and the cholangiocarcinoma cell lines(RBE,QBC939,HuH-28,and HuCCT1)with different degrees of differentiation were selected for validation.The para-carcinoma tissue and normal human intrahepatic biliary epithelial cell(HIBEC)were used as the control groups.The expression levels of lncRNA CASC2 and H19 in carcinoma tissue,para-carcinoma tissue,and cell lines were determined by real-time quantitative polymerase chain reaction(qRT-PCR).The correlation analysis was carried out for the clinical indicators of patients with the expression levels of the target genes. Results The two target genes showed significantly different expression between carcinoma tissue and para-carcinoma tissue(all P<0.05).Specifically,CASC2 had higher expression level in the carcinoma tissue than in the para-carcinoma tissue(t=1.262,P=0.025),whereas the expression of H19 showed an opposite trend(t=1.285,P=0.005).The expression levels of CASC2 in QBC939(t=8.114,P=0.015)and HuH-28(t=9.202,P=0.012)cells were significantly higher than that in the control group.The expression levels of H19 were significantly lower in RBE(t=-10.244,P<0.001),QBC939(t=-10.476,P<0.001),HuH-28(t=-19.798,P<0.001),and HuCCT1(t=-16.193,P=0.004)cells than in the control group.Bioinformatics analysis showed that CASC2 was mainly involved in the metabolic process and H19 in the development of multicellular organisms.Both CASC2 and H19 were related to catalytic activity.The expression level of lncRNA CASC2 was correlated with pathological differentiation(χ 2=6.222,P=0.022)and lymph node metastasis(χ2=5.455,P=0.020),and that of lncRNA H19 with pathological differentiation(χ2=1.174,P=0.029)and tumor size(χ2=-0.507,P=0.037). Conclusions In the case of ECC,lncRNA CASC2 and H19 have transcription disorders.lncRNA CASC2 is generally up-regulated in the carcinoma tissue,while H19 is down-regulated.Both genes have the potential to become new molecular markers for ECC.

LncRNA SNHG3 Facilitates the Malignant Phenotype of Cholangiocarcinoma Cells via the miR-3173–5p/ERG Axis

BackgroundLong noncoding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) is an oncogenic lncRNA that has been reported in many cancers, but the role of SNHG3 in cholangiocarcinoma (CCA) remains largely unknown. Bioinformatic analysis revealed a regulatory relationship among SNHG3, miR-3173–5p, and ERG. miR-3173–5p is a tumour suppressive miRNA, while ERG is an oncogene. In the present study, we focused on the regulatory effects and molecular mechanisms of SNHG3 in CCA. MethodThe expression of SNHG3 and miR-3173–5p was evaluated using qRT–PCR analysis. Knockdown of SNHG3 was achieved by shRNA. Cell viability was assessed by MTT assay. Migration and invasion were determined by Transwell assay. Flow cytometry was used to assess cell apoptosis. Western blots were applied to quantify protein levels. Furthermore, using RNA pulldown and dual luciferase assays, the interactions between SNHG3 and miR-3173–5p and between miR-3173–5p and ERG in CCA cells were validated. ResultsSNHG3 was significantly upregulated in CCA cells compared with normal human intrahepatic biliary epithelial cells. Knockdown of SNHG3 inhibited the proliferation and migration of CCA cells. Mechanistically, SNHG3-sponged miR-3173–5p, thus releasing the repression of ERG by miR-3173–5p. Rescue experiments showed that the miR-3173–5p/ERG axis mediated the oncogenic effect of SNHG3. ConclusionTaken together, our data suggest that SNHG3 is a pleiotropic oncogenic lncRNA in CCA. Knockdown of SNHG3 expression suppressed malignant phenotypes in CCA cells via the miR-3173–5p/ERG axis.

Effect of lncRNA PVT1/miR186/KLF5 Axis on the Occurrence and Progression of Cholangiocarcinoma.

This study primarily focused on the effect of the long noncoding RNA (lncRNA) PVT1/miR186/KLF5 axis on the occurrence and progression of cholangiocarcinoma (CCA). miR186 was found both in the lncRNA PVT1 targeting miRNAs and KLF5 targeting miRNAs using bioinformatic analysis. The expression of lncRNA PVT1 and KLF5 in the TFK-1, QBC939, and HuCCT1 cell lines and normal biliary epithelial HIBEpiC cells was detected by RT-qPCR. The significance of lncRNA PVT1 and KLF5 on cell proliferation was analyzed using the MTT assay and clone formation assay in lncRNA PVT1 and KLF5 silencing HuCCT1 cell lines and lncRNA PVT1and KLF5 overexpressing TFK-1 and QBC939 cell lines, respectively. The potential role of lncRNA PVT1 and KLF5 in cell migration was detected using the transwell invasion assay in CCA cell lines and tumor formation assay. Additionally, lncRNA PVT1 and KLF5 were proved to be highly expressed in CCA tissues and cell lines. Silencing and overexpressing of lncRNA PVT1 or KLF5 markedly inhibited or increased the cell proliferation and cell invasion in CCA cell lines, respectively. Silencing and overexpressing of lncRNA PVT1 significantly inhibited and increased the expression of KLF5 in CCA cell lines, respectively. Silencing of lncRNA PVT1 increased the expression of miR186, and silencing of miR186 increased the expression of KLF5 in CCA cell lines. Cotransfection of lncRNA PVT1 and miR186 increased the expression of KLF5 compared with controls. Overall, these results demonstrated that the lncRNA PVT1/miR186/KLF5 axis might exert a key role in the occurrence and progression of CCA, and this axis might provide a new target for treating CCA.

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.

LncRNA TUG1 Promotes Growth and Metastasis of Cholangiocarcinoma Cells by Inhibiting miR-29a.

BackgroundAs a highly malignant tumor, cholangiocarcinoma poses a serious threat to human life and health, so exploring the mechanisms of its development and progression at a molecular level is of great significance to the diagnosis and treatment of the disease.ObjectiveThis study was aimed at investigating the effects and related mechanisms of LncRNA TUG1 on cholangiocarcinoma cells.MethodsCholangiocarcinoma tissues and adjacent tissues (n=82 each), human cholangiocarcinoma cell lines (RBE, QBC939, HuH28), and a human normal biliary epithelial cell line (HIBE) were collected. miR-29a-mimics, miR-29a-inhibitor, miR-NC, si-TUG1, pcDNA3.1 TUG1, and NC were transfected into the cholangiocarcinoma cells. qRT-PCR was performed to detect TUG1 and miR-29a expression in the cholangiocarcinoma tissues and cells. Western blotting (WB) was conducted to detect the expression of Bax, Caspase-3, and Bcl-2 in the cells. CCK-8 assay, Transwell, and flow cytometry were carried out to detect cell proliferation, invasion, and apoptosis. Dual luciferase reporter gene assay (DLRGA) was performed to confirm the correlation of TUG1 with miR-29a.ResultsTUG1 was highly expressed while miR-29a was poorly expressed in cholangiocarcinoma cells. TUG1 expression was negatively correlated with miR-29a expression, and TUG1 had a relatively high diagnostic value for cholangiocarcinoma. Cell experiments showed that inhibiting TUG1 expression or up-regulating miR-29a expression could inhibit cholangiocarcinoma cells from proliferation and invasion, and promote their apoptosis, while up-regulating TUG1 or inhibiting miR-29a could promote the proliferation and invasion but inhibit the apoptosis. Rescue experiment showed that overexpressing miR-29a could reverse the effects of high TUG1 expression on cholangiocarcinoma cells. DLRGA confirmed that there was a regulatory relationship between TUG1 and miR-29a.ConclusionTUG1 is highly expressed in cholangiocarcinoma tissues. It can promote the growth and metastasis of cholangiocarcinoma cells by inhibiting miR-29a, so it may be a new target for diagnosing and treating cholangiocarcinoma.

Effects of lncRNA MEG3 on proliferation and apoptosis of gallbladder cancer cells through regulating NF-κB signaling pathway.

To explore the effects of long non-coding ribonucleic acid (lncRNA) maternally expressed gene 3 (MEG3) on proliferation and apoptosis of gallbladder cancer (GBC) cells and its molecular mechanism. The relative expression level of lncRNA MEG3 in GBC cell lines was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). The lncRNA MEG3 expression plasmids were constructed, the cell proliferation ability was detected via Cell Counting Kit-8 (CCK-8) assay and colony formation assay, and the apoptosis was detected via flow cytometry. The effects of lncRNA MEG3 expression on endoplasmic reticulum stress (ERS)-related proteins were determined using Western blotting, and the changes in nuclear factor-κB (NF-κB) protein in the nucleus were determined after overexpression of lncRNA MEG3. The expression of lncRNA MEG3 in three kinds of GBC cell lines was lower than that in human immortalized normal biliary epithelial cells (p<0.05). The results of CCK-8 assay and colony formation assay showed that overexpression of lncRNA MEG3 significantly reduced the proliferation rate and colony formation ability of GBC-SD cells compared with negative control (NC) group (p<0.05, p<0.05). According to the results of flow cytometry, the apoptosis rate was higher in lncRNA MEG63 overexpression group compared with that in NC group (p<0.05). Moreover, the ERS-related proteins (MANF, GRP78, and caspase-3) were remarkably upregulated in lncRNA MEG63 overexpression group compared with those in NC group, indicating that ERS is activated by lncRNA MEG63 overexpression. The NF-κB signal in GBC cells was activated by lncRNA MEG3. LncRNA MEG3 activates the NF-κB signal in GBC cells to affect the proliferation and apoptosis of GBC cells.

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.

Notch1 Drives the Formation and Proliferation of Intrahepatic Cholangiocarcinoma.

The molecular mechanisms underlying the development of intrahepatic cholangiocarcinoma (ICC) are not clear yet. In this study, we investigated the involvement of Notch1 in the development of ICC. The cDNA microarray analysis showed that Notch1 expression was higher in ICC tissues than in normal biliary epithelial cells. Stable transfection of Notch1 receptor intracellular domain (NICD1) by hydrodynamic tail vein injection induced ICC formation in mice. Western blotting confirmed that Notch1 signaling was activated in human ICC cell lines and mouse ICC tissues. Silencing Notch1 with specific short interfering RNA (siRNA) inhibited the proliferation of ICC cells. Flow cytometry and Western blotting indicated that apoptosis was induced in Notch1-silenced ICC cells compared with controls. Additionally, Notch1 silencing was associated with the inhibition of hairy and enhancer of split-1 (Hes1) and activation of the phosphatase and tensin homolog (PTEN)/p53 pathway. Taken together, these data suggest that Notch1 drives ICC formation and proliferation; downregulation of Notch1 induces apoptosis in ICC cells; Notch1 signaling may serve as a novel therapeutic target for the treatment of ICC.

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.

Tumor-specific Expression of Insulin-like Growth Factor II mRNA-binding Protein 3 Independently Predicts Worse Survival of Patients With Adenocarcinoma of the Ampulla of Vater.

Insulin-like growth factor II mRNA-binding protein 3 (IMP3) plays an important role in the adhesion, invasion, and metastasis of tumor cells. Although emerging evidence suggests that IMP3 promotes tumor progression in several malignancies, the expression of IMP3 and its prognostic implication in adenocarcinoma of the ampulla of Vater (AVAC) has not been clarified to date. The IMP3 expression status in 87 AVAC tissues was examined using immunostaining, and its association with various clinicopathological features and outcome of patients with AVAC was investigated. The vast majority (87.4%) of AVAC cases displayed at least focal cytoplasmic and membranous IMP3 immunoreactivity in tumor cells, whereas IMP3 expression was consistently absent from normal biliary epithelial cells. Tumor-specific IMP3 expression was associated with submucosal and pancreatic invasion, which were not identified in the corresponding hematoxylin and eosin-stained slides. This finding led to up-staging of the pathological tumor stage in two cases of well-differentiated AVAC. In addition, high IMP3 expression was significantly associated with a poorly differentiated histology (p=0.026). Survival analyses revealed that high IMP3 expression independently predicted shorter recurrence-free (p=0.003) and overall (p=0.029) survival. Our study demonstrated tumor-specific IMP3 expression in AVAC, which will be helpful in determining invasion depth and tumor extent in patients with well-differentiated tumors, as well as indicating worse survival of patients with AVAC. Our data highlight IMP3 expression status as a potential diagnostic and prognostic marker for AVAC.

Elevation of circular RNA circ_0005230 facilitates cell growth and metastasis via sponging miR-1238 and miR-1299 in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly malignant carcinoma with high mortality rate worldwide. Emerging evidence indicates that aberrantly expressed circular RNAs (circRNAs) functions crucial roles in tumor progression. In this work, we focused on a novel circRNA, circ_0005230, in carcinogenesis and development of CCA. Circ_0005230 levels in CCA specimens and cells were measured by qRT-PCR. The clinical implication of circ_0005230 was analyzed by fisher’s exact test. Gain/loss of-function assays were conducted to reveal the effects of circ_0005230 on the cell proliferation, apoptosis, migration and invasion of CCA cells. Xenograft and lung metastatic models were constructed to confirm the in vitro data. Dual luciferase reporter and rescue assays were carried out to illuminate the mechanism behind the regulatory actions. As data showed, circ_0005230 was elevated in tumors and CCA cells. Its expression in tumor samples was related to clinical severity. Functionally, circ_0005230 significantly facilitated cell growth, clone-forming ability and metastatic properties and inhibit cell apoptosis in CCA cells. The in vivo study further validated the in vitro results. However, knockdown of circ_0005230 did not affect normal biliary epithelial (HIBEC) cell growth and apoptosis. For the mechanism investigation, circ_0005230 could directly sponge miR-1238 and miR-1299 to exert its oncogenic functions. Overall, this work showed that circ_0005230 might act as an effective therapeutic target for CCA.

Establishment of Patient-Derived Organoids and Drug Screening for Biliary Tract Carcinoma

Biliary tract carcinomas (BTCs) are among the most aggressive malignancies and have a poor prognosis. Here, we successfully established organoid lines derived from intrahepatic cholangiocarcinoma, gallbladder cancer, and neuroendocrine carcinoma of the ampulla of Vater. These organoids derived from BTCs were cultured stably for >1 year and closely recapitulated the histopathology, gene expression, and genetic alterations evident in the primary tumors. Gene expression profiling of the organoids revealed that SOX2 could be a potential prognostic biomarker for patients with BTC. We screened a compound library consisting of drugs used clinically for their ability to suppress organoids derived from BTCs and found that the antifungal drugs amorolfine and fenticonazole significantly suppressed the growth of organoids derived from BTCs with minimal toxicity to normal biliary epithelial cells. Patient-derived organoids may be a powerful research tool for the clarification of molecular pathogenesis and the discovery of biomarkers and therapeutic drugs for refractory cancers.

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.

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.

C‑Myc promotes cholangiocarcinoma cells to overcome contact inhibition via the mTOR pathway.

The loss of contact inhibition is a hallmark of a wide range of human cancer cells. Yet, the precise mechanism behind this process is not fully understood. c‑Myc plays a pivotal role in carcinogenesis, but its involvement in regulating contact inhibition has not been explored to date. Here, we report that c‑Myc plays an important role in abrogating contact inhibition in human cholangiocarcinoma (CCA) cells. Our data show that the protein level of c‑Myc obviously decreased in contact-inhibited normal biliary epithelial cells. However, CCA cells sustain high protein levels of c‑Myc and keep strong proliferation ability in confluent conditions. Importantly, the suppression of c‑Myc by inhibitor or siRNA induced G0/G1 phase cell cycle arrest in confluent CCA cells. We demonstrate that the inhibition of c‑Myc suppressed the activity of mammalian target of rapamycin (mTOR) in confluent CCA cells, and mTOR inhibition induced G0/G1 phase cell cycle arrest in confluent CCA cells. In confluent CCA cells, the activity of Merlin is downregulated, and Yes-associated protein (YAP) sustains high levels of activity. Furthermore, YAP inhibition not only induced G0/G1 phase cell cycle arrest, but also decreased c‑Myc expression in confluent CCA cells. These results indicate that Merlin/YAP/c‑Myc/mTOR signaling axis promotes human CCA cell proliferation by overriding contact inhibition. We propose that overriding c‑Myc‑mediated contact inhibition is implicated in the development of CCA.

Expression of sal-like 4 in primary hepatocellular carcinoma and its association with epithelial-mesenchymal transition

Objective: To investigate the expression of sal-like 4 (SALL4) in tissues of primary hepatocellular carcinoma (HCC) and its association with epithelial-mesenchymal transition (EMT) and clinicopathological characteristics. Methods: Immunohistochemistry and nucleic acid in situ hybridization were used to measure the mRNA expression of SALL4, epithelial cadherin (E-cadherin), vimentin, and Snail in 72 HCC samples, 2 fetal liver samples, and 2 normal adult liver tissue samples. Results: Strong expression of SALL4 was observed in hepatoblasts in fetal liver, but SALL4 expression was not observed in primitive hematopoietic cells and normal adult hepatocytes or biliary epithelial cells. In the HCC samples, the positive rate of SALL4 was 47.2% (34/72), showing focal positive nuclear staining. The HCC patients with microscopic microvascular tumor thrombus and portal vein tumor thrombus, a serum alpha-fetoprotein level of≥ 350 ng/ml, International Union Against Cancer (UICC) stage III+IV, and an age of < 46 years showed higher positive expression of SALL4 than those with no microscopic microvascular tumor thrombus or portal vein tumor thrombus, a serum alpha-fetoprotein level of < 350 ng/ml, UICC stage I+II, and an age of≥46 years. The HCC patients with positive SALL4 showed lower postoperative disease-free survival rate and overall survival rate than those with negative SALL4 (P < 0.05). Sixty percent of the patients with microvascular tumor thrombus (21/35) showed positive expression of SALL4. The positive rate of SALL4 was negatively correlated with E-cadherin (r = -0.434, P < 0.01), but positively correlated with vimentin and Snail (vimentin: r = 0.516, P < 0.01; Snail: r = 0.571, P < 0.01). Conclusion: In patients with primary HCC, the expression of SALL4 greatly affects EMT, which helps with the research on invasion and metastasis of liver cancer and prognostic evaluation.