QBC939 Cells Research Articles

Fragile histidine triad (FHIT) suppresses proliferation and promotes apoptosis in cholangiocarcinoma cells by blocking PI3K-Akt pathway.

Fragile histidine triad (FHIT) is a tumor suppressor protein that regulates cancer cell proliferation and apoptosis. However, its exact mechanism of action is poorly understood. Phosphatidylinositol 3-OH kinase (PI3K)-Akt-survivin is an important signaling pathway that was regulated by FHIT in lung cancer cells. To determine whether FHIT can regulate this pathway in cholangiocarcinoma QBC939 cells, we constructed an FHIT expression plasmid and used it to transfect QBC939 cells. Protein and mRNA expression were measured by western blotting and qRT-PCR, respectively. The viability and apoptosis of QBC939 cells were then assessed using MTT assays and flow cytometry. Our results revealed that the expression of survivin and Bcl-2 was downregulated, and caspase 3 was upregulated, in cells overexpressing FHIT. In addition, FHIT suppressed the phosphorylation of Akt. The changes in cell proliferation and apoptosis were obvious in cells overexpressing FHIT which parallels that of treatment with LY294002, a potent inhibitor of phosphoinositide 3-kinases. Treatment with LY294002 further decreased the expression of survivin and Bcl-2 and increased caspase-3 levels. These results suggest that FHIT can block the PI3K-Akt-survivin pathway by suppressing the phosphorylation of Akt and the expression of survivin and Bcl-2 and upregulating caspase 3.

Si-DNMT1 restore tumor suppressor genes expression through the reversal of DNA hypermethylation in cholangiocarcinoma

The aim of our study was to evaluate the effect of shorthairpin RNA plasmid vector knockdown of human DNA methyltransferase 1 on proliferation and the methylation status and expression of tumor suppressor genes in hilar cholangiocarcinoma. The hilar cholangiocarcinoma cell line QBC939 was utilized for this study. QBC939 cells were transfected with a shorthairpin RNA plasmid vector targeting human DNA methyltransferase 1. Control and human DNA methyltransferase 1 shorthairpin RNA plasmid vector-transfected cells were collected at different time points, and the expression levels of human DNA methyltransferase 1 and tumor suppressor genes (cyclin-dependent kinase inhibitor 2B, cyclin-dependent kinase inhibitor 2A, RAS association domain family 1, and cadherin-1) were detected by reverse transcription-polymerase chain reaction. Furthermore, interfering efficiency was confirmed by Western blotting. The methylation status of tumor suppressor genes was detected using methylation-specific polymerase chain reaction. Furthermore, the effect of human DNA methyltransferase 1 knockdown on proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Targeted gene knockout of human DNA methyltransferase 1 restored the expression levels of tumor suppressor genes cyclin-dependent kinase inhibitor 2B, cyclin-dependent kinase inhibitor 2A, RAS association domain family 1, and cadherin-1, indicating that the silencing of these tumor suppressor genes is associated with promoter hypermethylation. In addition, knockdown of human DNA methyltransferase 1 expression significantly inhibited the proliferation of QBC939 cells. Targeted knockdown of human DNA methyltransferase 1 expression restores the expression levels of tumor suppressor genes, thus inhibiting the proliferation of QBC939 cells. These results may provide insight for the development of novel therapies for cholangiocarcinoma.

Protective Effects of Guava Pulp on Cholestatic Liver Injury.

Background. Cholestatic liver injury is a leading cause of chronic liver diseases involved with oxidative stress changes and inflammation; thus, antioxidant and anti-inflammation compound-rich guava may play a pivotal role in protecting against the cholestatic liver damages. Our aims for this study are to determine whether guava pulp (GP) has protective effects on cholestatic liver injury-induced mouse model and on interleukin-6 (IL-6) mediated proliferation of QBC939 cholangiocarcinoma cell line. Methods. Mice were induced to cholestatic liver damage by left and median bile duct ligation (LMBDL) surgery and then treated with GP. Plasma and liver samples were collected for biochemical and pathological assays. 5-Bromo-2′-deoxyuridine (BrdU) assay and Western blots were used to detect proliferation and gene expression in QBC939 cells, respectively. Results. Compared with LMBDL only group, in GP-treated mice, the levels of alanine aminotransferase (ALT) and bilirubin decreased, biliary epithelial cell proliferation and liver fibrogenesis were suppressed, Src/MEK/ERK1/2/c-Myc pathway and expressions of transforming growth factor β1(TGF-β1), tissue inhibitor of metalloproteinases TIMP), and procollagen 1α1(COL1α1) were downregulated significantly. Moreover, the GP extract reduced IL-6-enhanced QBC939 cell proliferation, p-ERK, and c-Myc expression as well. Conclusions. GP may provide a new perspective for the treatment of cholestatic liver injury.

Study of transfection of Livin antisense oligodeoxynucleotide on apoptosis of QBC939 cells and its mechanism

Objective To study the effect of transfection of Livin antisense oligodeoxynucleotide (Livin ASODN) on Livin mRNA and Livin protein expression and apoptosis of QBC939 cells.Methods Livin ASODN were transfected into cell line QBC939 byLipofectamine TM 2000.60 h After transfection,to measure Livin mRNA expression by reverse transcriptase polymerase chain reaction (RT-PCR) and Livin protein expression by immunohistochemistry and confocal laser scanning microscopy ; the changes of cell apoptotic were detected by methyl thiazol tetrazolium (MTT).and the apoptosis of QBC939 cell was detected by Annexin V-PE flow cytometry (FCM) after the transfection.Results The mRNA and protein expression level of Livin in ASODN group was significantly lower than that in control group (P ＜0.05) (54.96 ± 3.00,88.31 ± 2.50,85.90 ± 0.62,90.36 ± 2.43) ; the results of MTT showed that the rate of cell apoptosis of QBC939 cells were the most obvious at 60 hours after Livin ASODN transfection (46.41 ± 1.13,3.10 ±0.44,2.66 ± 0.26,0) (P ＜ 0.05).the apoptosis of QBC939 cell was higher than those in control group after Livin ASODN transfection [(36.98 ±4.09)％,(3.77 ±0.25)％,(7.51 ±0.3)％,(6.83 ±0.23) ％].Conclusion The transfection of Livin ASODN inhibited Livin gene and protein expression,and Obviously induced apoptosis of Bile duct cancer QBC939 cells,which may be the new target for gene therapy in Bile duct cancer. Key words: Livin; Antisense oligodeoxynucleotide; Bile duct cancer; Cell apoptosis

Aberrant expression of laminin γ2 correlates with poor prognosis and promotes invasion in extrahepatic cholangiocarcinoma

BackgroundTo investigate the potential role of laminin γ2 and its correlation with prognosis in patients with extrahepatic cholangiocarcinoma (CCA). Materials and methodsLaminin γ2 expression was evaluated by immunohistochemistry in 72 extrahepatic CCA patients after surgical resection. Knockdown of laminin γ2 was achieved via small interfering RNA transfection in the extrahepatic CCA cell line QBC939. ResultsThirty-six of 72 extrahepatic CCAs (50%) stained positive for laminin γ2 in two types of patterns: stromal staining (28/72, 39%) and cytoplasmic staining (24/72, 33%). All 16 paracancerous tissue samples showed negative staining. Both stromal and cytoplasmic laminin γ2 expressions correlated with lymph node metastasis. Kaplan-Meier analysis showed that aberrant expression of laminin γ2 correlated with poor overall survival and early recurrence. Cox regression analysis further demonstrated that laminin γ2 expression was a significant independent predictor of poor overall survival and early recurrence. Immunofluorescence staining revealed cytoplasmic expression of laminin γ2 in QBC939 cells. Knockdown of laminin γ2 significantly reduced QBC939 cell invasion and migration. ConclusionsAberrant expression of laminin γ2 correlates with poor prognosis and promotes invasion in extrahepatic CCA.

Inhibition of Wnt/β‐catenin signaling downregulates P‐glycoprotein and reverses multi‐drug resistance of cholangiocarcinoma

The development of multi-drug resistance (MDR) represents a major obstacle in the successful treatment of cancers. However, the factors and mechanisms that lead to MDR in cholangiocarcinoma (CCA), a chemoresistant bile duct carcinoma with a poor prognosis, remain unclear. In this study, we established a human MDR CCA cell line QBC939/5-FU. Compared with QBC939 cells, a rounder shape, a higher nuclear-cytoplasmic ratio, a shorter cell cycle, faster growth and resistance to chemotherapeutics are major characteristics of QBC939/5-FU cells. P-glycoprotein (P-gp) and β-catenin were upregulated in QBC939/5-FU cells. Furthermore, the drug susceptibility of QBC939 cells to common chemotherapeutics was significantly decreased after Wnt3a treatment, whereas inhibition of Wnt/β-catenin pathway by β-catenin siRNA reversed the MDR of QBC939/5-FU cells to chemotherapeutics. Molecular study revealed that activation of Wnt/β-catenin pathway resulted in upregulation of P-gp and contributed to MDR of QBC939/5-FU cells. Extraction of Siamese Crocodile 3 (ESC-3) bile enhanced the drug sensitivity of QBC939/5-FU cells to 5-FU, paralleled with downregulation of β-catenin and P-gp. The association of Wnt/β-catenin pathway and P-gp was further confirmed by the clinical data for CCA tissues. Our study represents the first implication of Wnt/β-catenin activation in the MDR of CCA, which may be a beneficial target for the clinical treatment of CCA.

MicroRNA differential expression profile in cholangiocarcinoma cell line QBC939 and normal bile duct cell line HIBEC

Objective To explore microRNA differential expression profile between cholangiocarcinoma cell line QBC939 and normal bile duct cell line HIBEC.Methods Six samples from QBC939 cell line and another 6 samples from normal bile duct cell line HIBEC were chosen as the experimental group and the control group,respectively.MicroRNA profiles in these samples were analyzed by microarray.The threshold value used to screen up-and down-regulated microRNAs was fold chang ＞ 2.Real-time quantitative polymerase chain reaction (Real-time PCR) was used to validate the reliability of microarray.Results Six microRNAs were up-regulated in the experimental group and were more than 8 times higher than in the control group.Five microRNAs were down-regulated and were more than 8 times higher than in the control group.Conclusion These microRNAs with differential expression might be related with the formation and metastasis of cholangiocarcinoma. Key words: Cholangiocarcinoma; MicroRNA

Photosan-II loaded hollow silica nanoparticles: Preparation and its effect in killing for QBC939 cells

Nanoparticles have been explored recently as an efficient means to deliver photosensitizers for photodynamic therapy. However, it is largely unknown if polyhematoporphyrin (C34H38N4NaO5, Photosan-II, PS) or other photosensitizers can be efficiently delivered by hollow silica nanoparticles (HSNP). Polyhematoporphyrin (C34H38N4NaO5, Photosan-II, PS) was loaded into hollow silica nanoparticles (HSNP) by one-step wet chemical-based synthetic route. Dynamic light scattering (DLS) and polydispersive index (PDI) were used for measurement of the particles size and size distribution. Transmission electron microscope and scanning electron microscopy were used for the microstructure, morphological and chemical composition analysis. Fourier transform infrared spectrometry spectra and fluorescence emission spectrum were obtained. The photobiological activity of the PS-loaded HSNP was evaluated on human cholangiocarcinoma QBC939 cells. The cellular viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptotic and necrotic cells were measured by flow cytometry. DLS measurements showed that the size of the particles is in the range of 25-90 nm. PDI of the PS-loaded HSNP is 0.121 ± 0.01, indicating that samples have excellent quality with narrow size distribution to monomodal systems. In MTT assay, PS-loaded HSNP and free PS of the same concentration killed about 95.3% ± 2.0% and 55.7% ± 1.9% of QBC939 cells, respectively. The flow cytometry demonstrated that the laser induced cell death with PS-loaded HSNP was much more severe than that of free PS (P<0.05). Photosan-II-loaded hollow silica nanoparticles not only can quickly deliver Photosan-II into cells but also can reach a more high concentration than free Photosan-II. HSNP is a desirable vehicle and the release system that shows promises for photodynamic therapy use, which not only improve the aqueous solubility, stability and transport efficiency of PS, but also increase its photodynamic efficacy compared to free PS.

Over-Expression of Nerve Growth Factor-β in Human Cholangiocarcinoma QBC939 Cells Promote Tumor Progression

AimsIt has been shown that nerve growth factor-β (NGF-β) promoted the initiation and progression of many tumors, and we have previously demonstrated that the expression of NGF-β was associated with tumor stage, nerve infiltration and lymph node metastasis in human hilar cholangiocarcinoma. However, whether NGF-β promotes tumor progression in human cholangiocarcinoma requires further investigation. Therefore, we aimed to determine the effects of NGF-β on the progression of human cholangiocarcinoma.MethodsHuman cholangiocarcinoma QBC939 stable cell lines with over-expressed or silenced NGF-β genes were generated with pEGFP-N1-NGF-β and pGPU6/GFP/Neo-NGF-β-shRNA recombinant plasmids. Cell proliferation assay, colony formation assay, cell cycle analysis, apoptosis assay and tumorigenicity assay were performed to evaluate the role of NGF-β in the progression of human cholangiocarcinoma. In addition, human lymphatic endothelial cells were co-cultured with QBC939 culture supernatants, and the cell proliferation and migration abilities of the lymphatic endothelial cells were evaluated.ResultsForced expression of NGF-β in QBC939 cell lines promoted proliferation, colony formation and tumorigenicity in these cells and inhibited the apoptosis. However, down-regulation of NGF-β inhibited proliferation, colony formation and tumorigenicity, and increased the apoptotic rate of QBC939 cells. In addition, the NGF-β gain-of-function induced a high expression of vascular endothelial growth factor C and enhanced the proliferation and migration of lymphatic endothelial cells, while NGF-β loss-of-function showed opposite effects.ConclusionsWe concluded that NGF-β promoted tumor progression in human cholangiocarcinoma QBC939 cells. Our results provided a new concept to understand the role of NGF-β in cholangiocarcinoma progression, and might provide important information for the development of new targeted therapies in human cholangiocarcinoma.

Blockage of hedgehog signaling pathway inhibits the proliferation of QBC939 cholangiocarcinoma cell

Objective This article aims to study the impact of cyclopamine,a Hedgehog signaling pathway inhibitor,on the proliferation and apoptosis of QBC939 cholangiocarcinoma cells.Methods The proliferation of QBC939 cells was detected with the MTT assay,and the apoptotic rate was analyzed with the flow cytometry assay.RT-PCR and Western blow were used to detect the expressions of tumor-related genes and proteins in QBC939 cells before and after cyclopamine treatment.Results Our results show that cyclopamine inhibited the growth of QBC939 cells in time and dose dependent manners.After a 5,10,or 20 μmol/L cyclopamine treatment for 48 hours,QBC939 cells showed increased apoptotic rates significantly higher than those in the control group (P＜0.01).Furthermore,cyclopamine down regulated the mRNA and protein levels of PTCH1,GLI1,and EGFR in QBC939 cells.Conclusion Therefore,blockage of the Hedgehog signaling pathway with cyclopamine could suppress the proliferation and promote the apoptosis of QBC939 cholangiocarcinoma cells. Key words: Bile duct neoplasms; Cell proliferation; Apoptosis; Cyclopamine

Prognostic Significance of Capn4 Overexpression in Intrahepatic Cholangiocarcinoma

BackgroundCalpain small subunit 1 (Capn4) has been shown to correlate with the metastasis/invasion of hepatocellular carcinoma. This study aimed to investigate the role of Capn4 in intrahepatic cholangiocarcinoma (ICC).MethodsCapn4 expression was measured in 33 ICC tissues by quantitative real-time polymerase chain reaction and western blot. The role of Capn4 in the migration, invasion and proliferation of ICC cells and matrix metalloproteinase 2 (MMP2) expression were assessed after Capn4 depletion by specific small interfering RNA. Capn4 expression was further examined by immunohistochemistry in a tissue microarray consisting of 140 ICC patients and 13 normal liver tissues, and the prognostic role of Capn4 in ICC was evaluated by Kaplan-Meier and Cox regression analyses.ResultsCapn4 expression was significantly higher in the ICC tissues compared to the peritumor tissues. Capn4 down-regulation impaired the migration/invasion ability of HCCC-9810 and QBC939 cells in vitro and decreased MMP2 expression. Capn4 overexpression significantly correlated with the presence of lymphatic metastasis of ICC (p = 0.026) and the tumor-node-metastasis (TNM) stage (p = 0.009). The postoperative 2- and 5-year overall survivals in patients with Capn4low were higher than those in the Capn4high group. The cumulative recurrence rate in patients with Capn4low was much lower than in the Capn4high group. Multivariate analysis showed that Capn4 overexpression was an independent prognostic marker in ICC.ConclusionsCapn4 overexpression was implicated in ICC metastasis/invasion, and Capn4 overexpression may be used as a molecular therapeutic target for ICC.

Mucins 1‐shRNA inhibit the proliferation and HIF‐1alpha protein expression on human cholangiocarcinoma cells

To explore the effects of Mucins (MUC)1-shRNA on the proliferation and hypoxia inducible factor (HIF)-1alpha expression of human cholangiocarcinoma (CCA) QBC939 cells in vitro. MUC1-shRNA was constructed and transfected with Lipofectamine™ 2000 into cultured CCA cells. MUC1 mRNA and protein expression levels were determined by RT-PCR and Western blot, respectively. The cellular proliferation and HIF-1alpha expression of QBC939 cells were evaluated by the MTT assay and Western blot, respectively. After transfection, the expression levels of MUC1 mRNA and protein in the experimental group decreased significantly in QBC939 (P < 0.01). The proliferation of MUC1 shRNA-transfected group was 0.30 ± 0.05, 38.32 ± 1.43%, 15.18 ± 1.32%, and there were remarkable differences when compared with the control groups (P < 0.05). Significant inhibition of HIF-1alpha protein expression in MUC1 shRNA-transfected group was also discovered (P < 0.05). MUC1-shRNA could inhibit proliferation and significantly weaken HIF-1alpha protein expression of QBC939 cells, suggesting its potential as a therapeutic target of CCA.

Photodynamic therapy promotes apoptosis of cholangiocarcinoma QBC939 cells

Photodynamic therapy promotes apoptosis of cholangiocarcinoma QBC939 cells

Preparation, Characterization and Phototoxicity of Photosan-Loaded Hollow Silica Nanospheres for Photodynamic Therapy

In this study, Photosan (PS) was loaded onto hollow silica nanospheres using one-step wet chemical-based synthetic route method. Polydispersive index of the Photosan-loaded hollow Silica Nanospheres (PS-loaded NSs) is 0.121±0.01. The photobiological activity of the PS-loaded NSs was evaluated on human cholangiocarcinoma QBC939 cells. Cells were incubated with free PS or PS-loaded NSs for 2 h and then washed with phosphate-buffered saline. Culture medium was added to the wells containing the cells. Finally, the cells were exposed to red light (630 nm) with a light dose of 10J/cm2. The cellular viability was determined after 24 h of incubation. PS-loaded NSs and free PS eliminated about 96.4%±2.0% and 54.7%±1.9% of QBC939 cells, respectively. The phototoxicity was time dependent up to 2 h and concentration dependent at 1-7.5 mg/l. The cells viability decreased with the increase of the light dose in the range of 5-15 J/cm2. In conclusion, PS-loaded NSs are the release systems that promise photodynamic therapy use.

The Combined Effects of Hematoporphyrin Monomethyl Ether-SDT and Doxorubicin on the Proliferation of QBC939 Cell Lines

It is well established that both hematoporphyrin monomethyl ether-sonodynamic therapy (HMME-SDT) and doxorubicin (DOX) can induce cell apoptosis but each alone has its own limitations. To date, the combined effects of HMME-SDT and DOX on inducing cell apoptosis are little known and the mechanism for the combined effects remains poorly understood. In the present study, we reported the synergistic effects of HMME-SDT and DOX on inhibiting the proliferation of human cholangiocarcinoma QBC939 cells and investigated the mechanism of this synergy. The data from MTT assay, flow cytometer, Hoechst staining and cell arrest analysis showed that the combination of HMME-SDT and DOX exhibited higher inhibiting effects on proliferation of QBC939 cells than the sole application of HMME-SDT or DOX. In addition, the synergistic effects were shown to result from the DNA damage as demonstrated by single cell gel electrophoresis and DNA fragmentation. Furthermore, the expression of p53, Fas, Bax and activated caspase-3 protein was significantly upregulated in cells treated with HMME-SDT and DOX, whereas Bcl-2 protein was downregulated. Taken together, our data suggested that the application of HMME-SDT combined with DOX had better inhibiting effects on QBC939 cells and the effects were caused mainly by DNA damage.

Expression of carcinoembryonic antigen-related cell adhesion molecule 6 in human hilar cholangiocarcinoma cell line QBC939 and its role in invasion and metastasis

Objective To explore the expression of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) in human hilar cholangiocarcinoma cell line QBC939 and its roles in invasion and metastasis.Methods Real-time polymerase chain reaction (real-time PCR) was used to detect the expression of CEACAM6 in QBC939 cells.Lentiviral-mediated CEACAM6 small interfering RNA (siRNA) was established to infect QBC939 cells.The silencing effect was assayed by real-time PCR.The invasive activity of infected QBC939 cells was analyzed by Transwell invasion assay.Results There was high expression of CEACAM6 in QBC939 cells.Recombinant lentiviral vector expressing siRNA targeting CEACAM6 gene was established successfully and confirmed by PCR and DNA sequencing.The expression of CEACAM6 mRNA in QBC939 cells infected with LV-CEACAM6-siRNA was decreased by 93.1％ as compared with control group (P ＜ 0.05).After CEACAM6 gene was knocked down,Transwell test revealed that the invasion potency of QBC939 cells was significantly suppressed by 69.2％ as compared with control group (P ＜ 0.05).Conclusion There was high expression of CEACAM6 in QBC939 cells.There is a positive correlation between the expression level of CEACAM6 and the invasion potency of hilar cholangiocarcinoma. Key words: RNA interference; Carcinoembryonic antigen-related cell adhesion molecule 6; Hilar cholangiocarcinoma

Gamma-Aminobutyric Acid Binds to GABAB Receptor to Inhibit Cholangiocarcinoma Cells Growth via the JAK/STAT3 Pathway

Gamma-aminobutyric acid (GABA) has been reported to inhibit the growth of cholangiocarcinoma QBC939 cells, but the mechanisms are still not fully understood. To explore the mechanisms of the anti-cancer effect of GABA on QBC939 cells. An initial immunohistochemistry study of the expressions of GABA receptors in cholangiocarcinoma tissues was followed by the culture and treatment of QBC939 cells for 48 h with GABA, GABA + bicuculine (GABAA receptor antagonist), GABA + phaclofen (GABAB receptor antagonist), and GABA + AG490 (Janus Kinase inhibitor). MTT and Annexin V-FITC/PI binding assays were used to determine the proliferation and apoptosis of the QBC939 cells. The expression of the signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (Tyr705) [p-STAT3 (Tyr705)] was evaluated by the western blot assay. The effect of GABA on the growth of QBC939 xenograft tumors in athymic nu/nu mice was examined, and p-STAT3 (Tyr705) expression in xenograft tumors was detected by immunohistochemistry. A significant difference was only observed in GABAB receptor expression between cholangiocarcinoma and normal bile tissues. The MTT and Annexin V-FITC/PI assays showed that the antiproliferative and proapoptotic effects of GABA on QBC939 cells could be antagonized by phaclofen and AG490, but not bicuculine. GABA significantly down-regulated p-STAT3 (Tyr705) expression; this action was also antagonized by phaclofen and AG490. GABA also effectively inhibited xenograft tumor growth, and p-STAT3 (Tyr705) expression was significantly decreased in GABA-treated xenograft tumors. GABA may inhibit the growth of cholangiocarcinoma QBC939 cells through the GABAB receptor, and the anti-cancer effects may be partly mediated via the JAK/STAT3 pathway.

Identification of up-regulated miRNAs in extrahepatic and intrahepatic cholangiocarcinoma

Objective To investigate the expression profile of miRNAs up-regualted in human extrahepatic and intrahepatic cholangiocarcinoma tissues and probe the effect on cell growth of four of these miRNAs in QBC939 cell line.Methods Up-regulated miRNAs in extrahepatic or intrahepatic cholangiocarcinoma tissues were analyzed by using miRNA-microarray,which was confirmed by using miRNA Real-Time PCR analysis.Based on these findings,four of these up-regulated miNRAs were chosen to perform function investigation.The specific miRNA inhibitors were transfected into QBC939 cells,respectively,and cell proliferation assay was performed by using MTT.Results 12 miRNAs were up-regulated both in two types of cholangiocarcinoma tissues,28 miRNAs and 21 miRNAs were up-regulated in extrahepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma,respectively.MiR-125b and miR-19a expression levels were increased about 3.7 and 3.6 fold,compared with the matched normal bile duct tissues (P＜0.05).MiR-92a and miR-205 expression was upregulated about 4.S- and 3.5-fold,compared with the matched normal bile duct tissues (P＜0.05).MiR-125b,miR19a,miR-21,and miR 378* were inhibited in QBC939 cells,which indicated a significant inhibitory effect on cell growth.The ratio of inhibition was 71％,72％,69％,and 76％(P＜0.05)at 36 h,61％,63％,60％,and 59％(P＜0.01) at 48 h,and 61％、56％、60％ and 59％(P＜0.05) at 60 h.Conclusion The miRNAs expression patterns in human extrahepatic and intrahepatic cholangiocarcinoma tissues are different and uo-regulated miRNAs act as oncomirs on cholangiocarcinoma cell growth. Key words: Extrahepatic cholangiocarcinoma; Intrahepatic cholangiocarcinorna; MicroRNAs; Cell growth

Experimental study of emodin inducing apoptosis of bile duct carcinoma cell QBC939

Objective To investigate the effect and mechanism of emodin inducing the apoptosis of human bile duct carcinoma cell QBC939 in vitro. Methods The inhibition of cell proliferation was detected by MTT. The morphological changes of apoptotic cells were observed under fluorescence microscopy.The apoptosis rates and intracellular reactive oxygen species levels were detected by flow cytometry.The intracellular relative activities of caspase-9 and caspase-3 were tested through colorimetric method.Results Emodin inhibited cell proliferation of human bile duct carcinoma cell QBC939 in time-dose dependent fashion.Apoptotic cells displayed bright,nuclear presented divided leaves,debris and a set of edges under fluorescence microscopy.Treated with 30 μmol/L and 50 μmol/L emodin,the apoptosis rates of 24 hours were respectively 38.9％ ± 9.07％ and 67.09％ ± 4.08％ (P ＜ 0.05).The intracellular reactive oxygen species levels after 30 minutes treatment were 1.65 ± 0.08 and 2.28 ± 0.04folds of the control group (P ＜ 0.05). Emodin could activate caspase-9 and caspase-3,leading to elevations of their activities (P ＜ 0.05).Conclusion Emodin inhibites cell proliferation of human bile duct carcinoma cell QBC939 through inducing apoptosis.The mechanism is associated with the elevated levels of reactive oxygen species and the activation of caspase-9 cells and caspase-3. Key words: Emodin; Apoptosis; Reactive oxygen species; Caspases

Targeted Drug Regulation on Methylation of p53—BAX Mitochondrial Apoptosis Pathway Affects the Growth of Cholangiocarcinoma Cells

To study the mechanism of 5-aza-2-deoxycytidine (DAC; a methylation inhibitor) on growth of the human cholangiocarcinoma QBC939 cell line. A colourimetric assay was used to detect growth of QBC939 cells treated with DAC (0.1-100 μmol/l) over 24 h, 48 h and 72 h. Cell morphology was observed by transmission electron microscopy (TEM). The cell cycle and apoptosis were analysed by flow cytometry. Hypermethylation of the promoters of the p53-BAX mitochondrial apoptosis genes cyclin-dependent kinase inhibitor 2A (CDKN2A), death-associated protein kinase 1 (DAPK1) and PYD and CARD domain containing (PYCARD) was detected by methylation-specific polymerase chain reaction, with and without DAC treatment. DAC inhibited QBC939 cell growth with a half maximal inhibitory concentration of 5 μmol/l at 72 h. After DAC treatment, apoptosis was observed by TEM. Flow cytometric analysis of propidium iodide-positive cells demonstrated increased apoptosis of DAC-treated QBC939 cells (43.04%) compared with untreated cells (4.31%). DAC treatment resulted in demethylation of the gene promoters of CDKN2A and DAPK1 in QBC939 cells. DAC induces apoptosis of QBC939 cells by reactivation of hypermethylated p53-BAX mitchondrial apoptosis genes in cholangiocarcinoma cells.