QBC939 Cells Research Articles

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.

Expression of GLS1 in intrahepatic cholangiocarcinoma and its clinical significance.

Kidney‑type glutaminase (GLS1) plays a significant role in tumor metabolism. Our recent studies demonstrated that GLS1 was aberrantly expressed in hepatocellular carcinoma (HCC) and facilitated tumor progression. However, the roles of GLS1 in intrahepatic cholangiocarcinoma (ICC) remain largely unknown. Thus, the aim of this study was to evaluate the expression and clinical significance of GLS1 in ICC. For this purpose, combined data from the Oncomine database with those of immunohistochemistry were used to determine the expression levels of GLS1 in cancerous and non‑cancerous tissues. Second, a wound‑healing assay and Transwell assay were used to observe the effects of the knockdown and overexpression of GLS1 on the invasion and migration of ICC cells. We examined the associations between the expression of GLS1 and epithelial‑mesenchymal transition (EMT)‑related markers by western blot analysis. Finally, we examined the associations between GLS1 levels and clinicopathological factors or patient prognosis. The results revealed that GLS1 was overexpressed in different digestive system tumors, including ICC, and that GLS1 expression in ICC tissue was higher than that in peritumoral tissue. The overexpression of GLS1 in RBE cells induced metastasis and invasion. Moreover, the EMT‑related markers, E‑cadherin and Vimentin, were regulated by GLS1 in ICC cells. By contrast, the knockdown of GLS1 expression in QBC939 cells yielded opposite results. Clinically, a high expression of GLS1 in ICC samples negatively correlated with E‑cadherin expression and positively correlated with Vimentin expression. GLS1 protein expression was associated with tumor differentiation (P=0.001) and lymphatic metastasis (P=0.029). Importantly, patients with a high GLS1 expression had a poorer overall survival (OS) and a shorter time to recurrence than patients with a low GLS1 expression. Multivariate analysis indicated that GLS1 expression was an independent prognostic indicator. On the whole, the findings of this study demonstrated that GLS1 is an independent prognostic biomarker of ICC. GLS1 facilitates ICC progression and may thus prove to be a therapeutic target in ICC.

LncRNA‐MEG3 inhibits cell proliferation and invasion by modulating Bmi1/RNF2 in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a mortal cancer with gradually increasing incidences all over the world, whereas effective diagnosis and treatment for this disease are still lacking. As a classical long noncoding RNA (lncRNA), maternally expressed gene 3 (MEG3) has been reported to exhibit pivotal regulatory roles in the occurrence and development of various digestive system tumors. Nevertheless, the clinical relevance and biological function of MEG3 in CCA remain largely unclear. In this study, MEG3 expression was significantly downregulated in both CCA tissues and cells in comparison with that in nontumor controls, respectively, and this downexpression was prominently associated with advanced TNM stage, lymph node invasion, and poor survival. Moreover, decreased MEG3 was an independent forecaster of poor prognosis for CCA patients. Functionally, MEG3 overexpression inhibited CCA growth in vitro and in vivo. Enhanced MEG3 also suppressed migration and invasion of CCLP-1 and QBC939 cells by reversing epithelial-mesenchymal transition (EMT) process. On the contrary, the proliferation, metastasis, and EMT were facilitated via knocking down MEG3. In addition, the expression of B lymphoma Mo-MLV insertion region 1 (Bmi1) and RING finger protein 2 was impacted by gain or loss of MEG3, furthermore, the malignant processes induced by MEG3 knockdown were rescued by means of silencing Bmi1. These data suggested that MEG3 caused tumor suppressive effects partly through mediating polycomb repressive complex 1. Our findings elucidate that MEG3 exerts critical functions in CCA development and likely acts as a promising tumor indicator or intervention target for CCA.

Effect of long-chain non-coding RNA AL136359.1 on the proliferation and migration of cholangiocarcinoma cells by up-regulating the expression of AKR1B10

Objective To detect the expression of AL136359.1 in cholangiocarcinoma tissues and cells and analyze its effect on proliferation and migration of cholangiocarcinoma cells. Methods The expression of AL136359.1 in 8 cholangiocarcinoma tissues and 4 cholangiocarcinoma cell lines (QBC939, RBE, HCCC-9810 and TFK-1) was detected by real-time quantitative PCR (qRT-PCR). Transfection of plasmid carrying AL136359.1 in the cholangiocarcinoma cell line with the lowest expression level of AL136359.1 were used to increase the expression of AL136359.1, and the transfection efficiency was verified by qRT-PCR. qRT-PCR and Western blot were used to detect the expression of AKR1B10 at mRNA and protein levels. The cell counting kit (CCK-8) and transwell migration assay were used to detect the effect of high expression of AL136359.1 on the proliferation and migration ability of cholangiocarcinoma cells. Results The expression of AL136359.1 in cholangiocarcinoma tissues (0.41±0.05) was significantly lower than that in adjacent tissues (2.94±0.44, P<0.01). The expression of AL136359.1 in cholangiocarcinoma cells was significantly lower than that in human normal cholangioepithelial cells (P<0.01), and the QBC939 cells had the lowest expression (P<0.01). High expression of AL136359.1 caused an increase in the expression of AKR1B10 at mRNA and protein levels (P<0.01). High expression of AL136359.1 could significantly inhibit the proliferation and migration of cholangiocarcinoma cells (P<0.01). Conclusions The expression of AL136359.1 were decreased in cholangiocarcinoma tissues and cell line. The high expression of AL136359.1 could up-regulate the mRNA expression of AKR1B10 gene, which was involved in inhibiting the proliferation and migration of QBC939 cells. Key words: Bile duct neoplasms; RNA, long noncoding; Cell proliferation; Cell migration; In vitro

LncRNA FENDRR represses proliferation, migration and invasion through suppression of survivin in cholangiocarcinoma cells

ABSTRACTThis study was to investigate the biological function and underlying mechanisms of FENDRR in cholangiocarcinoma (CCA) cell proliferation, migration and invasion. FENDRR and survivin expression in CCA tissues or cell lines were measured by qRT-PCR. In QBC939 and HuCCTl cells, cell proliferation was detected by CCK-8, cell migration and invasion were using transwell assay. RNA pull-down and RIP assay were performed to determine whether FENDRR can combine with SETDB1 in CCA cell. The effect of SETDB1 on survivin and H3K9me1 expression in CCA cells were determined by western blotting. ChIP analysis was performed to analyze the combination of SETDB1 with survivin promoter in CCA cell. The effect of SETDB1 knockdown on survivin and H3K9me1 expression in CCA cells after transfection with FENDRR were determined by western blotting. The results showed that lncRNA FENDRR was downregulated in CCA tissues and cells, and was negatively correlated with survivin expression. Further investigation demonstrated that FENDRR represses CCA cell proliferation, migration and invasion through regulating survivin expression. FENDRR associated with SETDB1 and H3K9 to epigenetically silence survivin and then regulated cell proliferation, migration and invasion. These findings indicate an important role for FENDRR–survivin axis in CCA cell proliferation, migration and invasion, and reveal a novel epigenetic mechanism for survivin silencing. Our data indicated that FENDRR silences survivin via SETDB1-mediated H3K9 methylation, thereby represses CCA cell proliferation, migration and invasion.

Apatinib affect VEGF-mediated cell proliferation, migration, invasion via blocking VEGFR2/RAF/MEK/ERK and PI3K/AKT pathways in cholangiocarcinoma cell

BackgroundCholangiocarcinoma (CCA) is a form of cancer that easily aggress to contiguous structures. Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) are increased in majority species of cancers and suppress tumor progression by blocking VEGF/VEGFR2. Apatinib is a highly selective VEGFR2 antagonist which has inhibitive effect on antiapoptotic and cell growth in CCA. While, the effect of apatinib cell migration and invasion in CCA is still unknown.MethodsCCA cell lines QBC939 and TFK-1 were transfected with siKDR to establish the KDR function loss cell model, and recombined human VEGF (rhVEGF) protein was added into the culture medium to enhance the VEGF expression. RT-qPCR and western bloting were used to detect the mRNA and protein expression levels of VEGFR2 to investigate whether it was effectively repressed or activated with rhVEGF or apatinib treatment. Then, MTT, wound healing assay, and transwell matrix assay were applied to measure the effect of apatinib and rhVEGF on cell viability, migration and invasion, respectively.ResultsThe mRNA and protein expressions of VEGFR2 were significantly reduced with KDR RNAi in both QBC939 and TFK-1 cells, and rhVEGF treatment increased these expression levels (p < 0.05). Apatinib dramatically suppressed VEGF-mediated cell migration and invasion at the concentration of 100 nM treatment and significantly decreased the expression of metastasis-associated protein such as Slug, snail and MMP9. Moreover, all of these inhibiting effects of apatinib depended on the VEGFR2 existence. In addition, VEGFR2/RAF/MEK/ERK and PI3K/AKT signal pathways were enhanced by the introduction of rhVEGF, but were dramatically suppressed after the apatinib treatment.ConclusionApatinib inhibit VEGF-mediated cell migration and invasion in CCA cell lines via inhibiting the VEGFR2/RAF/MEK/ERK and PI3K/AKT pathways. It will be a potentially effective targeted drug for CCA.

Effects of propofol on human cholangiocarcinoma and the associated mechanisms.

Cholangiocarcinoma (CCA) is the most common type of biliary duct malignancy. Propofol is a fast-acting intravenous anesthetic, which also exerts an anti-cancer effect. The aim of the current study was to explore the effects of propofol on human CCA and the associated mechanisms in vitro. The results indicated that as concentration (0, 1, 5 and 10 µg/ml) of propofol and treatment time (24, 48 and 72 h) increased, the cell inhibition rate of human CCA QBC939 cells increased. Furthermore, treatment with various concentrations of propofol for 48 h resulted in a decrease in migration and invasion capacity in QBC939 cells. Propofol also induced the apoptosis of QBC939 cells and cell cycle arrest in G1 phase. Propofol treatment increased the expression level of Bax and decreased that of Bcl-2. In addition, the effects of propofol on gene expression were evaluated, including Wnt3α, β-catenin, Snail1 and c-myc in the Wnt/β-catenin signaling pathway. It was identified that as the concentration of propofol increased, the expression of these genes decreased. In conclusion, the current results indicate that propofol is a promising therapeutic agent for the treatment of CCA.

Overpression of miR-29b suppresses the proliferation and induces apoptosis of cholangiocarcinoma cells

To investigate the expression of miR-29b in cholangiocarcinoma and explore its effects on cell proliferation and apoptosis of cholangiocarcinoma cells. Real-time PCR was used to detect the expression of miR-29b in cholangiocarcinoma cells line QBC939 and cholangiocarcinoma tissues. The lentiviral vector LV-hsa-miR-29b and blank vector were constructed to infect QBC939 cells. MTT assay and cell clone formation assay were performed to assess the changes in the cell proliferation and clone formation, respectively; flow cytometry was employed to evaluate the effect of miR-29b overexpression on cell cycle and apoptosis. The expression of miR-29b was significantly down-regulated in QBC939 cells and cholangiocarcinoma tissues as compared with H-69 cells and normal tissues (P &lt; 0.01). Compared with the blank vector, the lentiviral vector LV-hsa-miR-29b caused significantly increased expression of miR-29b in QBC939 cells (P &lt; 0.01), which exhibited suppressed cell proliferation and clone formation (P &lt; 0.01 or 0.05), cell cycle arrest at the S phase (P &lt; 0.05), and significantly increased cell apoptosis (P &lt; 0.01). As a tumor-suppressing miRNA, miR-29b is down-regulated in cholangiocarcinoma, and its overexpression can suppress the proliferation and induce apoptosis of cholangiocarcinoma cells.

Study on the correlation of the effect of microRNA-218 through Survivin on cholangiocarcinoma cells

Objective To study the effect of microRNA-218 (miR-218) on the invasion of cholangiocarcinoma cells by Survivin. Methods Two cholangiocarcinoma cell lines (CCLP1) and (QBC939) were cultured, and transfected with miR-218 mimicss. Negative control group and blank control group were set up. The invasiveness was determined via BD Martigel basement membrane matrix gel invasion Chamber. 3 results were randomly selected from each group. The CCLP1 and QBC939 cells were co transfected with miR-218 mimic and pMIR-Survivin, miR-218 mimic and pMIR-Survivin-mut, respectively. 3 results were randomly selected from each group, and the luciferase activity was detected by enzyme labelling instrument, and the correlation between miR-218 and Survivin was discussed. Results The numbers of CCLP1 cells transfected with miR-218, cells in negative control group, and cells in NC group were (28.33±4.16), (72.00±8.72) and (74.33±4.04) cells, respectively; The numbers of QBC939 cells transfected with miR-218, cells in negative control group, and cells in NC group were (7.33±2.08), (22.00±4.36) and (22.00±4.00) cells, respectively. Results showed that transfection of miR-218 mimics can inhibit the invasion ability of cholangiocarcinoma (The P values of CCLP1 cell line and QBC939 cell line were 0.000, 0.004). Luciferase activity detection results showed that luciferase activity in QBC939 wild type miRNA-218 group was 1.489±0.079, luciferase activity in negative control group was 2.825±0.068, luciferase activity in NC group was 2.747±0.081, luciferase activity in QBC939 mutant miRNA-218 group was 3.078±0.138, luciferase activity in negative control group was 2.944±0.144, the luciferase activity of the NC group was 2.807±0.090, the luciferase activity of the CCLP1 wild type miRNA-218 group was 1.322±0.039, the luciferase activity of the negative control group was 2.362±0.129, the luciferase activity of the NC group was 2.368±0.154, the luciferase activity of the CCLP1 mutant miRNA-218 group was 2.202±0.073, and the negative control group luciferase activity was 2.274±0.250, the activity of luciferase in group NC was 2.202±0.073. The luciferase activity of the wild type 3’untranslated regions (3’UTR) of the reporter gene vector was significantly lower than that of the negative control group and the blank control group (The P values of CCLP1 cell line and QBC939 cell line were 0.000, 0.000). While there was no significant change in the mutation type. Conclusion MiR-218 inhibits the invasion of cholangiocarcinoma cells and plays a negative regulatory role in the development of cholangiocarcinoma. miR-218 can interact with Survivin 3’UTR directly. Thus miR-218 can have a negative regulatory effect on cholangiocarcinoma through the Survivin directly. Key words: Cholangiocarcinoma; MicroRNA-218; Survivin; Cell invasion

High promoter activity of cytokeratin-19 gene in cholangiocarcinoma.

Conditionally replicating adenoviral vectors constructed with tumor-specific promoters (TSPs) offers a viable tool for the treatment of cholangiocarcinoma. The aim of the present study was to investigate cholangiocarcinoma-specific TSPs that remain active in adenoviral constructs in gene therapy. The mRNA expressions of cyclooxygenase-2, cytokeratin-19 (CK19), mucin-1, midkine and telomerase reverse transcriptase were determined in human cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes using reverse transcription-quantitative polymerase chain reaction. The candidate promoters constructed in adenoviral vectors were analyzed for their activities in cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes using dual-luciferase reporter assays. The mRNA expression of CK19 was markedly higher in the QBC939 cell line, indicating specificity to cholangiocarcinoma. Moreover, the promoter activity of CK19 in the adenoviral vector in infected cholangiocarcinoma cells was found to be significantly stronger compared with that in infected primary human hepatocytes and cholangiocytes. CK19 may be implicated in the pathogenesis of cholangiocarcinoma, as demonstrated by the stronger activity of its promoter, as well as the higher expression of mRNA in tumor cells. Therefore, the use of the promoter sequence of the CK19 gene may represent a potential tool in cholangiocarcinoma-specific adenoviral gene therapy.

IGFBP7 is associated to prognosis and could suppress cell survival in cholangiocarcinoma

Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted protein and its expression was restrained in varied solid tumours, but there was no report about biological role of IGFBP7 in cholangiocarcinoma (CCA). Here, we found that high expression of IGFBP7 correlated to a better overall survival in CCA patients. To investigate the hypothetic antineoplastic activity of IGFBP7 in CCA, we induced overexpression of IGFBP7 in QBC939 and RBE cells, as well as knockdown in HCCC9810 cells. And the biological functions triggered by level changes of IGFBP7 were assessed, including proliferation, cell cycle distribution, apoptosis and invasion evaluation. Cell growth assessment showed that enhanced IGFBP7 expression significantly retarded proliferation rates of QBC939 and RBE cells while an enhancement was observed in IGFBP7-inhibited HCCC9810 cells. The inhibition of cell viability was induced via G2/M phase arrest and apoptosis. Both QBC939 and RBE cells possess highly invasive ability, and IGFBP7 overexpression attenuated their serious invasiveness by reversing their mesenchymal phenotype to endothelial signature. We next investigated the potential mechanism involving in IGFBP7-induced tumour suppression and found that increased expression of IGFBP7 resulted in decrease of IGF-IR, IRS-1 and phosphor-AKT protein levels, accompanied with elevation of phorsphor-p38MAPK. These results suggest that IGFBP7 might be related to CCA carcinogenesis and metastasis, which further implicates that IGFBP7 might become a prospective benchmark for CCA diagnosis and therapy.

Porphyrin mediated photodynamic therapy synergistically boosts the radiosensitivity of cholangiocarcinoma cells to ionizing radiation

Objective To explore the effect of low intensity of porphyrin mediated photodynamic therapy (PDT) on the radiosensitization in cholangiocarcinoma cells and its related mechanisms. Methods 2, 7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) probe was used to detect the intracellular reactive oxygen species (ROS) production; Cell viability was evaluated by cell counting kit-8 (CCK-8) assays. Flow cytometry was used to quantify the apoptotic level of cells; Clonogenic survival assay was performed to assess the radiosensitizing effect of the radiosensitizer; Apoptosis related proteins were detected by Western blotting. Results Low intensity of PDT does not lead to death of QBC939 cells, whereas provides an elevated ROS environment in the mitochondria. The increasing intracellular level of ROS is demonstrated to enhance the radiosensitivity of QBC939 cells with a supra-additive synergistic effect of 0 + 1 >1 . The apoptotic rates in the PDT + radiotherapy group and the radiotherapy alone group were (15.45±1.23)% and (6.35±0.84)% respectively (P=0.001). In addition, the low intensity of PDT achieves a high SER10 value of 1.59, which is much larger than that of the most popularly used radiosensitizers, cisplatin (1.27), and paclitaxel (1.32). The expression of phosphorylated RAC-alpha serine/threonine-protein kinase (p-Akt) and phosphorylated extracellular regulated protein kinases (p-ERK) proteins decreased more significant after PDT + radiotherapy when compared to the radiotherapy alone (p-AKT and p-ERK were declined by (53.6±14.2)% and (49.8±17.5)%, respectively). Conclusion Low intensity of PDT is an ideal method for radiotherapy sensitization and has great potential for clinical application. Key words: Cholangiocarcinoma; Photodynamic therapy; Radiosensitization; Apoptosis

The effect of microRNA-224 on the anti-apoptosis and proliferation promoting of cholangiocarcinoma cells in vitro

Objective To explore the regulatory mechanism of microRNA-224 (miR-224) in the apoptosis and proliferation of human cholangiocarcinoma (CC) cells and examine whether the pathway of miR-224-HOXD10-PAK4 exist in cc cells or not. Methods The expression of miR-224 in CC and adjacent normal tissue were measured by RT-PCR. QBC939 cells were chosen and divided into two groups: cell groups transducted with negative control viruses (NC group) and cell groups transducted with target gene miRNA-down virus (DOMN group). The rate of apoptosis and the number of colonies formed in QBC939 cell lines of DOWN group and NC group were counted. The expression of miR-224, API5, PAK4 and HOXD10 in QBC939 cell lines of DOWN group and NC group were measured by RT-PCR and western blotting. Results (1) MiR-224 expression was higher in CC tissues than that in normal bile duct tissues (P<0.05). (2) In QBC939 cells lines, miR-224 expression was lower in DOMN group than that in NC group. The clone formation of QBC939 cells in DOWN group was significantly lower than that of NC group. The apoptosis rate of QBC939 cells of DOWN group was significantly higher than that of NC group. (3)The expression of API5 and HOXD10 in QBC939 cells of DOWN group was significantly higher than that of NC group in the RT-PCR and western blotting experiments. (4) The expression of PAK4 in QBC939 cells of DOWN group was significantly lower than that of NC group in the RT-PCR and western blotting. Conclusions MiR-224 has anti-apoptotic effects and can promote the proliferation of QBC939 cells and may serve as an important regulator of QBC939 cell proliferation in vitro. There are miR-224-HOXD10-PAK4 signaling pathways in cholangiocarcinoma. Key words: Cholangiocarcinoma; microRNA-224 (miR-224); Cell apoptosis; Proliferation; Signal transduction, API5P1, PAK4, HOXD10

Deoxypodophyllotoxin induces cell cycle arrest and apoptosis in human cholangiocarcinoma cells.

Deoxypodophyllotoxin (DPT), a naturally occurring flavolignan, has a broad range of biological effects, including anti-inflammatory, anti-viral and anticancer properties. The present study investigated the anti-proliferative effect of DPT on human cholangiocarcinoma QBC939 and RBE cell lines and its underlying mechanisms of inducing cytotoxicity. MTT assays demonstrated that DPT inhibited the viability of the QBC939 and RBE cells in a dose and time-dependent manner. In addition, DPT treatment resulted in G2/M phase cell cycle arrest associated with the downregulation of Cyclin B and cyclin dependent kinase 1 and caused an increase in apoptosis that was confirmed by characteristic morphological changes. Apoptosis was accompanied by increasing B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein ratios and activated expression of caspase-3, -8 and -9. These findings suggested that DPT may be a novel anticancer agent against human cholangiocarcinoma.

The influence of dihexyl adipate combined with NS-398 on the apoptosis of bile duct cancer cells QBC939

The influence of dihexyl adipate combined with NS-398 on the apoptosis of bile duct cancer cells QBC939

Inhibiting the proliferation and metastasis of hilar cholangiocarcinoma cells by blocking the expression of vascular endothelial growth factor with small interfering RNA.

The aim of the present study was to investigate whether the proliferation and metastasis of hilar cholangiocarcinoma cells can be suppressed and whether apoptosis can be induced by small interfering RNA (siRNA) repression of vascular endothelial growth factor (VEGF). siRNA sequences targeting the VEGF gene were designed and the human hilar cholangiocarcinoma QBC939, HCCC-9810 and RBE cell lines were transfected with VEGF-siRNA plasmids for 48 h. Reverse transcription-quantitative polymerase chain reaction and western blotting measured the levels of VEGF-A, VEGF-C and matrix metalloproteinase 2 (MMP2) mRNA expression and protein content. The cell invasion potential was evaluated using the Transwell invasion and migration assay and the MTT assay was employed to detect the proliferation of hilar cholangiocarcinoma cells. Flow cytometry was used to quantify cell apoptosis and necrosis. Following the transfection of VEGF-siRNA, a significant reduction of mRNA and protein levels of VEGF-A, VEGF-C and MMP2 was observed in the hilar cholangiocarcinoma cells. The invasion, migration and proliferation of tumor cells were also notably decreased. The rate of tumor cell apoptosis was increased in the VEGF-siRNA group (15.42%) compared with the non-siRNA control (2.22%) and the negative control (2.71%) groups. It was concluded that blocking the expression of VEGF via VEGF-siRNA effectively inhibited the invasion, migration and proliferation, and induced apoptosis in hilar cholangiocarcinoma cells. These observations suggested that targeting VEGF with RNAi may be an effective therapeutic strategy for treating hilar cholangiocarcinoma.

The effect of docosahexaenoic acid combined with cyclooxygenase-2 selective inhibitor NS-398 on the apoptosis of bile duct cancer cells QBC939

Objective To investigate the effect of docosahexaenoic acid (DHA) combined with cyclooxygenase-2 (COX-2) selective inhibitor NS-398 on the apoptosis of cholangiocarcinoma QBC939 cells and the mechanism. Methods In vitro, cholangiocarcinoma QBC939 cells were treated with 0, 15, 30, 45, 60 and 75 μg/ml DHA with 0, 25, 50, 100, 150 and 200 μmol/L NS-398, respectively. The absorbances of the QBC939 cells were measured by CCK8 and its growth inhibition ratios were analyzed. Flow cytometry was applied to detect cell apoptosis. The level of β-catenin and COX-2 mRNA and protein were measured by real-time PCR, immunocytochemistry and enzyme-linked immunoadsordent assay, respectively. Results DHA combined with NS-398 could significantly suppress the growth of QBC939 cells (P<0.05). When the concentration of DHA went up to 45 μg/ml and NS-398 was 100 μmol/L, the relative growth inhibition rate of QBC939 cells was 90.0%. If the concentrations were increased, the result showed no significant differences. Furthermore, flow cytometry analysis indicated that DHA combined with NS-398 could induce QBC939 cells apoptosis at the early stage, and the apoptosis rate was significantly different between the experimental and control groups (P<0.01). Real-time PCR showed low β-catenin and COX-2 expression in QBC939 cells disposed by DHA combined with NS-398, and their expression were significantly different between the experimental and control groups (P<0.01). Immunocytochemistry and ELISA demonstrated that DHA combined with NS-398 could decrease β-catenin and COX-2 protein expression in QBC939 cells. Conclusion DHA combined with NS-398 induced apoptosis and inhibited proliferation of cholangiocarcinoma cells QBC939 in vitro through targeting β-catenin and COX-2. Key words: Docosahexaenoic acid (DHA); NS-398; Cholangiocarcinoma; Apoptosis; β-catenin; Cyclooxygenase-2 (COX-2)

Effect of microRNA-148b and microRNA-152 on proliferation and apoptosis of cholangiocarcinoma cells

Objective To investigate the microRNA (miRNA, miR)-148b and miR-152 expression and their biological functions in cholangiocarcinoma cells. Methods The expression levels of miR-148b and miR-152 in cholangiocarcinoma simples and QBC939 cells were detected using real-time quantitative polymerase chain reaction (Real-time PCR). After transfection with miR-148b/miR-152 mimics, cell proliferation, cell cycle and apoptosis were assessed using methyl thiazol tetrazolium (MTT) method, clone formation test and flow cytometry, respectively. Results The expression levels of miR-148b and miR-152 were significantly down-regulated [(2.75±0.16) folds and (3.05±0.18) folds] in cholangiocarcinoma samples as compared with normal samples (P=0.013, 0.019, respectively), and [(2.52±0.03) folds and (3.96±0.02) folds] in QBC939 cells as compared with H-69 cells (P=0.018, 0.012, respectively). Overexpression of miR-148b or miR-152 could significantly inhibit cell proliferation [(38.49±0.31)% and (52.69±1.01)%, respectively], and suppress cell clone formation (P=0.027, 0.017, respectively), block cell cycle in the S phase and induce cell apoptosis. Conclusion MiR-148b and miR-152 were down-regulated in cholangiocarcinoma cells, and overexpression of miR-148b and miR-152 could inhibit cell proliferation and induce cell apoptosis. Key words: MicroRNA-148b; MicroRNA-152; Cholangiocarcinoma; Proliferation; Apoptosis

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.

PF-2341066 combined with celecoxib promotes apoptosis and inhibits proliferation in human cholangiocarcinoma QBC939 cells.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with high incidence and an average age of onset of 50-70 years old. However, at present there is no effective treatment for this disease. The aim of the present study was to investigate the effects of a c-Met inhibitor, PF-2341066 and a cyclooxygenase-2 (COX-2) inhibitor, celecoxib, on c-Met and COX-2 expression, proliferation and apoptosis. The results demonstrated that c-Met and COX-2 are highly expressed in hepatobiliary calculus with cholangiocarcinoma. PF-2341066 was able to downregulate the expression of c-Met and COX-2 in a dose-dependent manner at the mRNA and protein levels in human cholangiocarcinoma QBC939 cells. Furthermore, combined treatment with PF-2341066 with celecoxib downregulated the mRNA expression of both genes, inhibited cell proliferation and promoted cell apoptosis. It was also demonstrated that PF-2341066 and celecoxib treatment was able to restrict the expression of vascular endothelial growth factor (VEGF). The results of the present study suggest that PF-2341066 and celecoxib may inhibit the development of cholangiocarcinoma by downregulating the expression of c-Met and COX-2 to inhibit cell proliferation, promote apoptosis and prevent VEGF-mediated tumor angiogenesis. Co-treatment with PF-2341066 and celecoxib may be a potential therapeutic strategy for hepatobiliary calculus with cholangiocarcinoma.