CCA Tissues Research Articles

Long noncoding RNA SNHG6 promotes proliferation and angiogenesis of cholangiocarcinoma cells through sponging miR-101-3p and activation of E2F8.

Cholangiocarcinoma (CCA) development is an extremely complex process with alterations occurring in numerous genes. SNHG6, a validated lncRNA, has been reported to regulate the expression of multiple tumor-related genes in hepatocellular carcinoma, colorectal cancer and breast cancer. Here, we elucidated the function and possible molecular mechanisms of SNHG6 in human CCA cells. Our results proved that the expression SNHG6 was upregulated in CCA tissues and cell lines. Ectopic expression of SNHG6 promoted cell proliferation, cell cycle progression, migration, and angiogenesis in CCA cells, whereas knockdown of SNHG6 repressed these cellular processes. Further mechanistic studies revealed that SNHG6 could compete with the transcription factor E2F8 to bind with miR-101-3p, thus affecting E2F8 expression. Taken together, these results provided a comprehensive analysis of the role of SNHG6 in CCA cells and offered important clues to understand the key roles of competing endogenous RNA (ceRNA) mechanisms in human cholangiocarcinoma.

MiR-30a-5p promotes cholangiocarcinoma cell proliferation through targeting SOCS3.

Background: MicroRNAs (miRNAs) play important roles in the occurrence and development of cancers. In this project, we aimed to explore the role and molecular mechanism of mir-30a-5p in cholangiocarcinoma (CCA).Materials and Methods: The expression profile and clinical significance of miR-30a-5p in CCA patients were investigated in 31 ICC and 52 ECC patients respectively. The role and mechanism of miR-30a-5p in CCA cells were investigated by up-regulating and inhibiting miR-30a-5p expression in vitro functional study.Results: The expression of miR-30a-5p was increased in both CCA tissues and cells. The inhibition of miR-30a-5p decreased cell proliferation and induced cell apoptosis while overexpression of miR-30a-5p achieved the opposite effect. Furthermore, SOCS3 was down-regulated in ICC and ECC tissues and negatively regulated by miR-30a-5p. Dual-luciferase reporter assay revealed that co-transfection of miR-30a-5p significantly inhibited the activity of firefly luciferase reporter carrying the wild-type 3′UTR of SOCS3. The inhibition of SOCS3 could largely rescue the inhibitory effect of miR-30a-5p inhibition on CCA cells proliferation. In clinical, up-regulated miR-30a-5p expression was correlated with large tumor size in both ICC and ECC cohorts.Conclusions: miR-30a-5p promoted CCA cells proliferation through targeting SOCS3. These findings suggested that miR-30a-5p could be a potential therapeutic target.

Upregulation of DPY30 promotes cell proliferation and predicts a poor prognosis in cholangiocarcinoma

ObjectivesModification of lysine 4 on histone H3 methylation by SET1 and MLL family methyltransferase complexes is tightly linked to cancer progression. DPY30 is an important subunit of SET1 and MLL complexes, however, its expression and roles in cancer progression was little known, especially in cholangiocarcinoma (CCA). Materials and MethodsThe Q-PCR and IHC were performed to detect the levels of DPY30 mRNA and protein in CCA tissues. Effect of DPY30 knockdown on the proliferation of CCA cells was detected by MTS and colony formation, and cell cycle distribution was analyzed by flow cytometer. The glucose uptake, lactate release and ATP production assays were performed to detect the glycolysis of CCA cells. ResultsThe level of DPY30 mRNA and protein in CCA tissues were all significantly higher than that of pericancer tissues, and its upregulation was closely associated with pathological differentiation, tumor size, and TNM stage. In addition, Kaplan-Meier analysis of overall survival revealed that DPY30 upregulation was significantly associated with poor survival, and univariate and multivariate analysis indicated that it was an independently prognosis factor in CCA patients. Moreover, DPY30 knockdown inhibited in-vitro growth and induced cell cycle arrest at G2/M and decreased glycolysis in CCA cells. ConclusionsDPY30 upregulation may promote the development of CCA and was associated with the aggressive malignant behavior and poor survival outcome of CCA patients. DPY30 might serve as a potential novel target for treatment of CCA patients.

Serum coiled-coil domain containing 25 protein as a potential screening/diagnostic biomarker for cholangiocarcinoma.

Coiled-coil domain containing 25 (CCDC25) was previously reported to be upregulated in cholangiocarcinoma (CCA) tissues compared with adjacent normal tissues. The present study investigated whether serum CCDC25 level may be used as a potential marker for the diagnosis of CCA. Bioinformatics tools were used to reveal that CCDC25 is secreted into plasma/serum via a non-conventional pathway, which secretes proteins independently from the endoplasmic reticulum/golgi complex, but is yet to be fully elucidated. Subsequently, the CCDC25 levels in the sera of patients with CCA (n=141), patients with benign biliary disease (BBD; n=53) and healthy controls (HC; n=72) were measured using a quantitative dot blot assay based on the standard curve created using recombinant CCDC25 protein. The results demonstrated that the serum CCDC25 level in the CCA group (0.28±0.06 ng/µl) was significantly higher compared with that in the BBD (0.15±0.03 ng/µl) or HC (0.0017±0.0008 ng/µl) groups. Serum CCDC25 level provided an improved resolution (P=0.0001) compared with carcinoembryonic antigen (P=0.098) or carbohydrate antigen 19-9 (P=0.271) for the differential diagnosis between BBD and CCA. Receiver operating characteristic curve analysis revealed high sensitivity and specificity of serum CCDC25 level to differentiate between patients with CCA and HC (93.0 and 100%, respectively), and also to differentiate between patients with CCA and patients with BBD (75.0 and 84.0%, respectively). CCDC25 expression was further investigated in 23 CCA tissues, and CCDC25 expression in cancer tissues was moderately correlated with the serum CCDC25 level (r2=0.52, P=0.01). Among patients with CCA, serum CCDC25 level was significantly higher in patients with non-metastatic CCA compared with patients with metastatic CCA. Correspondingly, a higher serum CCDC25 level was associated with a longer overall survival time in patients with CCA. In conclusion, serum CCDC25 level may be a promising screening and diagnostic marker for the differential diagnosis of CCA.

Increase of MAL-II Binding Alpha2,3-Sialylated Glycan Is Associated with 5-FU Resistance and Short Survival of Cholangiocarcinoma Patients.

Background and objectives: Sialylation plays important roles in tumor progression. Our present study aimed to demonstrate the alteration of sialylation and its role in cholangiocarcinoma (CCA). Materials and Methods: The α2,3- and α2,6-sialylation in CCA tissue was analyzed by lectin-histochemistry using Maackia amurensis lectin-II (MAL-II) and Sambucus nigra agglutinin (SNA). CCA cell lines were treated with the pan-sialylation inhibitor 3Fax-peracetyl-Neu5Ac (3F-Sia) followed by proliferation and chemosensitivity assays. Results: MAL-II binding α2,3-Sialylated Glycan (MAL-SG) and SNA binding α2,6-Sialylated Glycan (SNA-SG) were both elevated in CCA compared with hyperplastic/dysplastic (HP/DP) and normal bile ducts (NBD). The positive staining for MAL-SG or SNA-SG were found in 82% (61/74) of the CCA cases. Higher expression of MAL-SG in CCA was associated with shorter survival of the patients. The median survival of patients with high and low MAL-SG were 167 and 308 days, respectively, with overall survival of 233 days, suggesting the involvement of MAL-SG in CCA progression. MAL-SG expression of CCA cell lines was markedly decreased after treatment with 3F-Sia for 48 to 72 h. While proliferation of CCA cells were not affected by 3F-Sia treatment, their susceptibility to 5-fluorouracil (5-FU) was significantly enhanced. These results suggest that sialylation is involved in the development of 5-FU resistance and the sialylation inhibitor 3F-Sia can be used as a chemosensitizer for CCA. Conclusions: Sialylation is critically involved in the development of chemoresistance of CCA, and sialylation inhibitors may be used as a chemosensitizer in CCA treatment.

CD147 augmented monocarboxylate transporter-1/4 expression through modulation of the Akt-FoxO3-NF-κB pathway promotes cholangiocarcinoma migration and invasion.

Cholangiocarcinoma (CCA) is an aggressive type of cancer. The major obstacles for treatment are its late presentation and the occurrence metastases. Targeting the metastatic process may serve as a treatment option. CD147 is a membrane protein that promotes CCA metastasis. High lactate levels in CCA are predicted to result from lactate dehydrogenase A expression and sensitivity to monocarboxylate transporter (MCT) inhibitors. An involvement of CD147 in MCT maturation has been reported, but the exact role of MCT in CCA is not clear. Here, we aimed to assess the mechanism of CD147-promoted CCA progression through MCT regulation. The expression levels of CD147 and MCT-1/4 in human CCA tissues were determined by immunohistochemistry. Two CD147 knockout (CD147 KO) CCAcell (KKU-213) clones were established using the CRISPR/Cas9 system. Cell migration and invasion were determined using a Boyden chamber assay. Temporal protein levels were modified by siRNA, specific inhibitors and/or activators. The expression of target proteins was determined using Western blot analyses. CD147 and MCT-1/4 were found to be overexpressed in CCA tissues compared to normal bile duct tissues. In addition, we found that CD147 knockdown significantly alleviated CCA cell migration and invasion, concomitant with decreased pAkt, pFoxO3, pNF-κB (pp65) and MCT-1/4 levels. Conversely, we found that FoxO3 knockdown led to recovered migration/invasion abilities and increased pp65 and MCT-1/4 expression levels. The involvement of Akt in the regulation of MCT-1/4 expression through CD147 was established by inhibition and activation of Akt phosphorylation. Our data indicate that CD147 promotes the malignant progression of CCA cells by activating the Akt-FoxO3-NF-κB-MCT-1/4 axis. As such, CD147 may serve as a possible target for advanced CCA treatment.

Receptor-Interacting Protein Kinase 1 Promotes Cholangiocarcinoma Proliferation And Lymphangiogenesis Through The Activation Protein 1 Pathway.

PurposeReceptor-interacting protein kinase 1 (RIPK1) is an important upstream regulator of multiple cell signaling pathways including inflammatory signals. RIPK1 is reported to be closely associated with the prognostic implications of cancer, especially epithelial tumors. But its role in proliferation and lymphangiogenesis in cholangiocarcinoma (CCA) remains unclear and requires further investigation.Patients and methodsExpression of RIPK1 in human CCA tissues and CCA cell lines (QBC939, HUH28 and CCPL-1) was measured using qPCR, immunoblotting and immunohistochemistry. Silencing of RIPK1 was achieved by transduction of CCA cells via lentiviral plasmids (LV3-H1/GFP&Puro) encapsulating RIPK1 shRNA (LV-shRIPK1) or negative control shRNA (LV-shNC), and puromycin was used to select stable colonies. Proliferation and lymphangiogenesis were assessed in vitro by CCK-8 and matrigel-based tube formation assays, respectively. Activity of the activation protein-1 (AP-1) was evaluated by double-luciferase reporter gene assay. Protein expression of JNK, P38MAPK, ERK1/2, AP-1, P-AP-1, E-cadherin, N-cadherin and vascular endothelial growth factor-C (VEGF-C) was measured by immunoblotting or ELISA. An orthotopic CCA model in null mice was generated by transplanting QBC939 LV-shRIPK1, LV-shNC and control cells to further evaluate the role of RIPK1 on lymphangiogenesis in vivo. Immunohistochemistry was utilized to evaluate the expression of RIPK1 and VEGF-C, and tumor lymphatic vessels in the CCA model mice.ResultsUpregulated expression of RIPK1 in CCA tissues was closely related to tumor size, lymph node metastasis and poor prognosis. RIPK1 promoted proliferation and lymphangiogenesis in CCA cells, and regulated the activation of JNK and P38MAPK-mediated AP-1/VEGF-C pathway. Finally, in vivo animal experiments in the orthotopic CCA mouse model further confirmed the function of RIPK1 in lymphangiogenesis.ConclusionThis is the first report demonstrating the role of RIPK1 in proliferation and lymphangiogenesis through the MAPK (JNK and P38MAPK)- AP-1 pathway in CCA.

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.

Upregulation of miR‑132‑3p in cholangiocarcinoma tissues: A study based on RT‑qPCR, The Cancer Genome Atlas miRNA sequencing, Gene Expression Omnibus microarray data and bioinformatics analyses.

MicroRNAs (miRNAs/miRs) have been reported to be closely associated with numerous human diseases, including cholangiocarcinoma (CCA). However, the number of miRNAs known to be involved in CCA is limited, and the association between miR-132-3p and CCA remains unknown. In the present study, the clinical role of miR-132-3p and its potential signaling pathways were investigated by multiple approaches. Reverse transcription-quantitative PCR (RT-qPCR), CCA-associated Gene Expression Omnibus (GEO), ArrayExpress and Sequence Read Archive (SRA) miRNA-microarray or miRNA-sequencing data were screened, and meta-analyses were conducted, in order to calculate the receiver operating characteristic (ROC) curve and standardized mean difference (SMD). The predicted target genes of miR-132-3p were obtained from 12 online databases and were combined with the downregulated differentially expressed genes identified in the RNA-sequencing data of CCA. Gene Ontology annotation and pathway analysis were performed in WebGestalt. Protein-protein interaction analyses were conducted in STRING. The Cancer Genome Atlas (TCGA) mRNA expression profiles were used to validate the expression levels of hub genes at the mRNA level. The Human Protein Atlas was used to identify the protein expression levels of hub genes in CCA tissues and non-tumor biliary epithelium. The meta-analyses comprised 10 groups of RT-qPCR data, eight GEO microarray datasets and one TCGA miRNA-sequencing dataset. The SMD of miR-132-3p in CCA was 0.75 (95% CI: 0.25, 1.24), which indicated that miR-132-3p was overexpressed in CCA tissues. This finding was supported by a summary ROC value of 0.80 (95% CI: 0.76, 0.83). The pooled sensitivity and specificity were 0.81 (95% CI: 0.59, 0.93) and 0.71 (95% CI: 0.58, 0.81), respectively. The relative expression level of miR-132-3p in the early stage of CCA (stages I–II) was 6.8754±0.5279, which was markedly lower than that in the advanced stage (stages III–IVB), 7.3034±0.3267 (P=0.003). Consistently, the miR-132-3p level in low-grade CCA (grades G1-G2) was 6.7581±0.5297, whereas it was 7.1191±0.4651 in patients with high-grade CCA (grades G3-G4) (P=0.037). Furthermore, 555 potential target genes of miR-132-3p in CCA were mainly enriched in the ‘Focal Adhesion-PI3K-Akt-mTOR-signaling pathway’. In conclusion, upregulation of miR-132-3p may serve a pivotal role in the tumorigenesis and progression of CCA by targeting different pathways. Further in vitro and in vivo studies are required to support the current findings.

Five Hub Genes Can Be The Potential DNA Methylation Biomarkers For Cholangiocarcinoma Using Bioinformatics Analysis.

BackgroundCholangiocarcinoma (CCA) is a subtype of highly malignant hepatic tumor, which has low 5-year survival rate and poor clinical outcome. Only a few patients can be detected early and accepted with the surgery. Most of CCA patients are diagnosed in advanced stage, and the treatments are limited. As for the inoperable, advanced CCA patients, chemotherapy is the main treatment, due to lacking molecular targets, therapeutic effect is limited.Materials and methodsTo explore potential therapeutic targets for CCA, we analyzed three microarray datasets derived from the Gene Expression Omnibus (GEO) database. Then, we used GEO2R tools of NCBI to discover the differentially expressed genes (DEGs) from the CCA and normal liver tumor microarrays (TMA). Subsequently, we used the Database for Annotation, Visualization and Integrated Discovery (DAVID GO) to perform the Gene Ontology function (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Then, we carried out the Cytoscape software to search for the hub genes downregulated in CCA and identify the protein–protein interaction (PPI) of these genes. Besides, we used the GEPIA tool to evaluate the differential expressions of hub genes in CCA patients. Then, we also used MEXPRESS database to detect the promoter methylation levels of hub genes in CCA and normal tissue samples. In addition, we evaluated the expression of these genes in CCA lines and normal bile tract cells after 5-AZA (DNA methyltransferase inhibitor) treatment.ResultsA total of 115 downregulated DEGs were identified. Among them, 10 hub genes with a high degree of connectivity were picked out. Among these 10 hub genes, F2, AHSG, ALDH8A1, SERPIND1 and AGXT showed higher DNA methylation levels of promoter in CCA compared with normal liver tissues. Therefore, these 5 genes may be the potential DNA methylation biomarkers and therapeutic targets in CCA.

Long non-coding RNA MEG3 represses cholangiocarcinoma by regulating miR-361-5p/TRAF3 axis.

The aim of this study was to investigate the effect of long non-coding RNA MEG3 (MEG3) and microRNA-361-5p (miR-361-5p) on cholangiocarcinoma cells and to explore the molecular mechanisms. The level of MEG3 and miR-361-5p was detected using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between miR-361-5p and MEG3/TNF receptor-associated factor 3 (TRAF3) was confirmed by the Dual-Luciferase Reporter Assay. 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry analysis were used to determine cell viability and cell apoptosis. Moreover, the protein level of TRAF3, p65, and p-p65 was measured by Western blot assay. We found that MEG3 was downregulated in CCA tissues and cell lines, especially in TFK1 and QBC939 cells. MEG3 directly bind to miR-361-5p, which was highly expressed in CCA tissues and cell lines. Further analysis indicated that MEG3-plasmid could inhibit cell viability and induce cell apoptosis in CCA cells, but these effects were significantly reversed by miR-361-5p mimic. Moreover, we proved that TRAF3 was a direct target of miR-361-5p and it was downregulated in CCA tissues and cell lines. In addition, we found that miR-361-5p downregulation significantly inhibited CCA cell viability and induced cell apoptosis, and these effects were eliminated by the knockdown of TRAF3. Further functional analysis showed that the knockdown of TRAF3 upregulated the expression of p-p65 decreased by miR-361-5p inhibitor in CCA cells. Our results suggested that MEG3 repressed cholangiocarcinoma by downregulating miR-361-5p expression. Meanwhile, the suppression of miR-361-5p might improve CCA survival by targeting TRAF3 and inhibiting the NF-κB pathway, which might help to develop new strategies for CCA therapy.

Urine proteomics study reveals potential biomarkers for the differential diagnosis of cholangiocarcinoma and periductal fibrosis.

Cholangiocarcinoma (CCA) is a primary malignant tumor of the epithelial lining of biliary track associated with endemic Opisthorchis viverrini (Ov) infection in northeastern Thailand. Ov-associated periductal fibrosis (PDF) is the precancerous lesion for CCA, and can be detected by ultrasonography (US) to facilitate early detection. However, US cannot be used to distinguish PDF from cancer. Therefore, the objective of this study was to discover and qualify potential urine biomarkers for CCA detection in at-risk population. Biomarker discovery was conducted on pooled urine samples, 42 patients per group, with PDF or normal bile duct confirmed by ultrasound. After depletion of high abundance proteins, 338 urinary proteins were identified from the 3 samples (normal-US, PDF-US, CCA). Based on fold change and literature review, 70 candidate proteins were selected for qualification by multiple reaction monitoring mass spectrometry (MRM-MS) in 90 individual urine samples, 30 per group. An orthogonal signal correction projection to latent structures discriminant analysis (O-PLS-DA) multivariate model constructed from the 70 candidate biomarkers significantly discriminated CCA from normal and PDF groups (P = 0.003). As an independent validation, the expression of 3 candidate proteins was confirmed by immunohistochemistry in CCA tissues: Lysosome associated membrane glycoprotein 1 (LAMP1), lysosome associated membrane glycoprotein 2 (LAMP2) and cadherin-related family member 2 (CDHR2). Further evaluation of these candidate biomarkers in a larger cohort is needed to support their applicability in a clinical setting for screening and monitoring early CCA and for CCA surveillance.

Overexpression of miR-25 is associated with progression and poor prognosis of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a rare but highly aggressive type of malignancy. MicroRNA (miR)-25 has been demonstrated to be involved in the genesis of numerous cancer types. The aim of the present study was to investigate the prognostic value and functional role of miR-25 in CCA. The expression of miR-25 was determined by reverse transcription-quantitative (RT-q)PCR. The association between miR-25 expression and clinicopathological features was analyzed using the χ2 test. Kaplan-Meier survival analysis and Cox linear regression were performed to explore the prognostic value of miR-25. The effects of miR-25 on the biological behavior of CCA cells were determined using loss-and gain-of-function experiments in CCA cell lines. Upregulated miR-25 expression was observed in CCA tissues and cell lines compared with that in the respective controls (all P<0.05). Patients with high expression of miR-25 in CCA tissues had a comparatively higher tumor-nodes-metastasis stage (P=0.026), a higher rate of lymph node metastasis (P=0.032) and a shorter overall survival rate (log-rank P=0.022). miR-25 was determined to be an independent prognostic factor for CCA patients (P=0.036). In vitro, transfection with miR-25 inhibitor suppressed cell viability, migration and invasion, while miR-25 mimics had the opposite effect. These results indicated that miR-25 functions as an oncogene and is involved in tumor progression in CCA. miR-25 may serve as a prognostic biomarker and a potential therapeutic target for CCA treatment.

CDK7 inhibitor THZ1 inhibits MCL1 synthesis and drives cholangiocarcinoma apoptosis in combination with BCL2/BCL-XL inhibitor ABT-263

Cholangiocarcinoma (CCA) is a fatal disease without effective targeted therapy. We screened a small-molecule library of 116 inhibitors targeting different targets of the cell cycle and discovered several kinases, including Cyclin-dependent kinase 7 (CDK7) as vulnerabilities in CCA. Analysis of multiple CCA data sets demonstrated that CDK7 was overexpressed in CCA tissues. Further studies demonstrated that CDK7 inhibitor THZ1 inhibited cell viability and induced apoptosis in CCA cells. We also showed that THZ1 inhibited CCA cell growth in a xenograft model. RNA-sequencing followed by Gene ontology analysis showed a striking impact of THZ1 on DNA-templated transcriptional programs. THZ1 downregulated CDK7-mediated phosphorylation of RNA polymerase II, indicative of transcriptional inhibition. A number of oncogenic transcription factors and survival proteins, like MCL1, FOSL1, and RUNX1, were repressed by THZ1. MCL1, one of the antiapoptotic BCL2 family members, was significantly inhibited upon THZ1 treatment. Accordingly, combining THZ1 with a BCL2/BCL-XL inhibitor ABT-263 synergized in impairing cell growth and driving apoptosis. Our results demonstrate CDK7 as a potential target in treating CCA. Combinations of CDK7 inhibition and BCL2/BCL-XL inhibition may offer a novel therapeutic strategy for CCA.

Novel tumor suppressor role of miRNA-876 in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a rare, highly invasive malignancy, and its incidence is increasing globally. MicroRNAs (miRNAs) mediate a wide array of cellular and biological processes and are dysregulated in various tumors. The functional and biological roles of miRNAs in CCA have not been fully elucidated. In this study, we show that miR-876 expression levels and copy number are significantly attenuated in the TCGA cohort of CCA tissue samples. TCGA expression data was consistent with the observed substantial decrease in miR-876 expression in patient samples and CCA cell lines. In-silico algorithm databases revealed BCL-XL as a potential target of miR-876. We observed miR-876 expression to be downregulated, whereas, BCL-XL upregulated in CCA cell lines. BCL-XL was identified as a direct functional target of miR-876 in CCA. miR-876-mediated reduction of BCL-XL regulated cell survival, induced apoptosis and caspase 3/7 expression in CCA. BCL-XL overexpression reversed the miR-876 mediated effect on CCA cell growth and apoptosis. Stable overexpression of miR-876 produced potent tumor suppressor activity and in vivo tumor cell growth reduction. Overexpression of miR-876 in a patient-derived xenograft (PDX) cell line significantly suppressed BCL-XL expression and spheroid formation with a concomitant induction of caspase 3/7 activity and apoptosis. This study demonstrates a novel tumor suppressor role for miR-876 in CCA, identifies BCL-XL as an actionable target, and suggests a potential therapeutic role for miR-876 in CCA.

Long non-coding RNA NNT-AS1 functions as an oncogenic gene through modulating miR-485/BCL9 in cholangiocarcinoma.

IntroductionGrowing evidence suggests that long non-coding RNAs (lncRNAs) could function as important regulators in carcinogenesis and cancer progression. Nicotinamide nucleotide transhydrogenase antisense RNA 1 (lncRNA NNT-AS1) is up-regulated in some human tumors and functions as a tumor promoter. This study aimed to detect the effect of NNT-AS1 on cholangiocarcinoma (CCA) prognosis.Materials and methodsIn this study, we detected NNT-AS1 expression in CCA tissue samples and cell lines, and analyzed the association between NNT-AS1 expression levels and clinical parameters of CCA patients. Moreover, we conducted loss-of-function studies in CCA cancer cells to explore the biological function and molecular mechanism of NNT-AS1. NNT-AS1 was downregulated by using RNAi technology. Cell proliferation was examined by CCK8 and clone formation assays. Cell migration and invasion were determined by wound healing and transwell assays. Western blot assays were used to explore protein expression.ResultsIn this study, NNT-AS1 was expressed at high levels in CCA and closely associated with poor prognosis of patients with CCA. NNT-AS1 knockdown impaired cell proliferation, suppressed CCA cell migration and invasion, and restrained tumor growth in vitro. Moreover, NNT-AS1 directly bounded to miR-485 and further regulated BCL9. Finally, rescue assays verified that NNT-AS1 modulated the tumorigenesis of CCA by regulating miR-485.ConclusionTaken together, NNT-AS1 played a critical biological role in the development of CCA. Our results elucidated NNT-AS1/miR-485/BCL9 axis might lead to a further understanding of the molecular mechanism of CCA.

Silencing of LAMC2 Reverses Epithelial-Mesenchymal Transition and Inhibits Angiogenesis in Cholangiocarcinoma via Inactivation of the Epidermal Growth Factor Receptor Signaling Pathway

Cholangiocarcinoma (CCA) is a malignant cancer that is associated with high mortality rates. The relationship between laminin γ 2 chain gene (LAMC2) and epidermal growth factor receptor (EGFR) has been previously documented in gastric cancer and oral squamous cell carcinoma. This study investigates the role of LAMC2 in epithelial-mesenchymal transition (EMT) and angiogenesis in CCA and explores the underlying mechanism(s). Differentially expressed genes related to CCA were initially screened using a microarray analysis, and the interaction between LAMC2 and the EGFR signaling pathway was identified. To determine the regulatory effects of LAMC2 on CCA progression, LAMC2 was silenced or overexpressed and the EGFR signaling pathway was activated or blocked. Subsequently, the regulation effects of LAMC2 were evaluated on the expression of EMT markers, invasion and migration of CCA cells, as well as microvessel density in nude mice. Microarray analysis demonstrated that highly expressed LAMC2 is linked to CCA development, which involves the EGFR signaling pathway. When LAMC2 expression was increased, the EGFR signaling pathway and EMT were activated in CCA tissues. Silencing of LAMC2 as well as EGFR signaling pathway inhibition led to suppression of EMT, cell invasion, and migration abilities invitro, as well as angiogenesis invivo. This study demonstrates that LAMC2 silencing suppresses the activity of the EGFR signaling pathway, thus functioning as a tumor suppressor in CCA.

Roles of Zinc Finger Protein 423 in Proliferation and Invasion of Cholangiocarcinoma through Oxidative Stress.

Zinc finger protein 423 (ZNF423) is a transcriptional factor involved in the development and progression of cancers but has not yet been examined in cholangiocarcinoma (CCA), an oxidative stress-driven cancer of biliary epithelium. In this study, we hypothesized that oxidative stress mediated ZNF423 expression regulates its downstream genes resulting in CCA genesis. ZNF423 protein expression patterns and 8-oxodG (an oxidative stress marker) formation in CCA tissues were investigated using immunohistochemical analysis. The results showed that ZNF423 was overexpressed in CCA cells compared to normal bile duct cells adjacent of the tumor. Notably, ZNF423 expression was positively correlated with 8-oxodG formation. Moreover, ZNF423 expression in an immortalized cholangiocyte cell line (MMNK1) was increased by hydrogen peroxide-treatment, suggesting that oxidative stress induces ZNF423 expression. To investigate the roles of ZNF423 in CCA progression, ZNF423 mRNA was silenced using specific siRNA in CCA cell lines, KKU-100 and KKU-213. Silencing of ZNF423 significantly inhibits cell proliferation and invasion of both CCA cell lines. Taking all these results together, the present study denoted that ZNF423 is an oxidative stress-responsive gene with an oncogenic property contributing to the regulation of CCA genesis.

LncRNA FLVCR1-AS1 regulates cell proliferation, migration and invasion by sponging miR-485-5p in human cholangiocarcinoma.

Long non-coding RNA (lncRNA) FLVCR1 antisense RNA 1 (FLVCR1-AS1) serves a crucial role in many types of cancer; however, to the best of our knowledge, the biological effect of FLVCR1-AS1 in cholangiocarcinoma (CCA) remains unclear. The present study aimed to elucidate the involvement of FLVCR1-AS1 in the regulation of human CCA cell growth, migration and invasion, as well as the mechanisms underlying its effect. The expression levels of FLVCR1-AS1 in CCA tumor tissues, adjacent normal tissues, CCA cell lines and a cholangiocyte cell line were determined by reverse transcription-quantitative polymerase chain reaction. A significantly higher expression level of FLVCR1-AS1 was identified in CCA tumor tissues and the CCA cell lines HuCCT1 and CCLP1 compared with the normal controls. Short hairpin RNA targeting FLVCR1-AS1 (shFLVCR1-AS1) and a control plasmid (shNC) were transfected into CCA cell lines. Cell proliferation, colony formation, migration and invasion of CCA cells transfected with shFLVCR1-AS1 were significantly suppressed compared with the shNC groups. The expression levels of migration and invasion-associated proteins, including Twist, matrix metalloproteinase (MMP)-2 and MMP-9, were also significantly suppressed by shFLVCR1-AS1-treatment. Furthermore, FLVCR1-AS1 knockdown inhibited tumor growth in a xenograft model. Mechanistically, FLVCR1-AS1 was demonstrated to sponge microRNA-485-5p (miR-485-5p) in human CCA. The expression of miR-458-5p was significantly decreased in CCA tissue compared with normal tissue, and Pearson's correlation analysis revealed that FLVCR1-AS1 expression was negatively correlated with miR-485-5p expression in CCA tissues. These results suggested that lncRNA FLVCR1-AS1 may be used as a novel therapeutic target and a potential diagnostic marker for CCA.

Dependency of Cholangiocarcinoma on Cyclin D–Dependent Kinase Activity

Cholangiocarcinoma (CCA) is a bile duct cancer with a very poor prognosis. Currently, there is no effective pharmacological treatment available for it. We showed that CCA ubiquitously relies on cyclin-dependent kinases 4 and 6 (CDK4/6) activity to proliferate. Primary CCA tissues express high levels of cyclin D1 and the specific marker of CDK4/6 activity, phospho-RB Ser780. Treatment of a 15-CCA cell line collection by pharmacological CDK4/6 inhibitors leads to reduced numbers of cells in the S-phase and senescence in most of the CCA cell lines. We found that expression of retinoblastoma protein (pRB) is required for activity of the CDK4/6 inhibitor, and that loss of pRB conferred CDK4/6 inhibitor-drug resistance. We also identified that sensitivity of CCA to CDK4/6 inhibition is associated with the activated KRAS signature. Effectiveness of CDK4/6 inhibition for CCA was confirmed in the three-dimensional spheroid-, xenograft-, and patient-derived xenograft models. Last, we identified a list of genes whose expressions can be used to predict response to the CDK4/6 inhibitor. Conclusion: We investigated a ubiquitous dependency of CCA on CDK4/6 activity and the universal response to CDK4/6 inhibition. We propose that the CDK4/6-pRB pathway is a suitable therapeutic target for CCA treatment.