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Cholangiocarcinoma (CCA) is one of the most malignant adenocarcinomas arising from bile duct epithelial cells. However, the molecular mechanism regulating CCA development and progression still needs to be investigated. Here we found that miR-365 was downregulated in CCA tissues compared with adjacent normal tissues. By functional experiments, we found that overexpression of miR-365 significantly inhibited CCA cell proliferation and promoted cellular apoptosis in vitro. Furthermore, administration with miR-365 markedly suppressed the growth of tumor tissues in vivo. Mechanistically, we identified E2F2 as the target gene of miR-365 in CCA cells. We found that overexpression significantly inhibited the expression of E2F2 in CCA cells, and there was an inverse correlation between the expression levels of E2F2 and miR-365 in CCA tissues. We also found that E2F2 was highly expressed in CCA tissues and cell lines. Restoration of E2F2 in miR-365-overexpressing CCA cells promoted cell viability and reduced cellular apoptosis in CCA. Collectively, our study demonstrated the essential role of miR-365 and its functional mechanism in CCA cells, which provided a new insight on the design of therapeutic targets for CCA treatment.

Deng-Yong Zhang,1 Zhong Liu,1 Zheng Lu,1 Wan-Liang Sun,1 Xiang Ma,1 Pei Zhang,2 Bin-Quan Wu,1 Pei-Yuan Cui1 1Department of General Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233000, Anhui, People’s Republic of China; 2Department of Pharmacy, Bengbu Medical College, Bengbu 233000, Anhui, People’s Republic of China Background: Cholangiocarcinoma (CCA) is a malignant tumor of the bile duct epithelium, including intrahepatic, perihilar, and distal CCA based on anatomical location. Hydroxysteroid dehydrogenase-like 2 (HSDL2) belongs to the SDR subfamily of oxidoreductases, and it is involved in glioma oncogenesis, as it can promote cell proliferation and inhibit cell apoptosis. The purpose of this study was to explore the underlying molecular mechanisms of HSDL2 in the process of CCA. Methods: HSDL2 expression levels were observed in CCA and adjacent (normal control) tissues by analyzing data from The Cancer Genome Atlas and Gene Expression Omnibus databases. A receiver operating characteristic curve analysis was carried out. In vitro, we overexpressed HSDL2 in RBE cells (a human CCA cell line) using a stable lentivirus-mediated transduction strategy. We then used quantitative real-time-PCR and Western blotting methods to detect the efficiency of HSDL2 overexpression. Cell proliferation was assessed using a Celigo Image Cytometer, MTT assays, and the expression of PCNA. Cell apoptosis was assessed by flow cytometry analysis, caspase3/7 activity, and the expression of the apoptotic markers BCL-2 and BAX. Results: We observed a downregulation of HSDL2 in CCA tissues based on The Cancer Genome Atlas and Gene Expression Omnibus data analysis. The receiver operating characteristic curve analysis showed that HSDL2 could be an excellent efficacy biomarker for CCA. In vitro, HSDL2 overexpression largely suppressed the proliferation of RBE cells. In addition, apoptosis was induced by HSDL2 overexpression. Conclusion: The results of the data analysis indicated that, compared with adjacent tissues, HSDL2 was downregulated in CCA tissues, and overexpressing HSDL2 in CCA cells suppressed growth and proliferation, which involved activating apoptosis. This helps to understand the underlying HSDL2-related molecular mechanisms in the process of CCA. Keywords: cholangiocarcinoma, CCA, HSDL2, lentivirus, proliferation, apoptosis

Background Many studies have shown that cinobufagin has antitumor effects against many cancers. The aim of this study was to assess the effects of cinobufagin on cholangiocarcinoma (CCA) cells. Methods Colony formation assay, cell-counting kit-8 (CCK-8) assay, and tumor xenograft experiments were applied to investigate the function of cinobufagin on human CCA cells, in vitro and in vivo. Flow cytometric analysis was performed to validate the effects of cinobufagin on cell apoptosis. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot assays were performed to evaluate expression levels of related genes. Results This study found that cinobufagin inhibited proliferation in both QBC939 and RBE cells. Flow cytometric analysis indicated that rate of apoptosis was significantly increased when treated with cinobufagin. Regarding mechanism analysis, cinobufagin was found to dramatically inactivate Notch signaling pathways. Animal experiment results in vivo were consistent with outcomes in vitro. Conclusions These data suggest that cinobufagin-mediated inactivation of Notch pathways may play an important role in the induction of apoptosis in CCA.

Cholangiocarcinoma (CCA) is a dismal cancer. At present, there is no effective chemotherapeutic regimen for CCA. This may be due to the marked resistance of CCA to chemotherapy drugs, for which a mechanism remains unknown. Nuclear factor-κB (NF-κB) is constitutively activated in a variety of cancer cells, including CCA. It has been shown to play roles in growth, metastasis, and chemoresistance of cancer. In the present study, we examined whether NF-κB is involved in the chemoresistance of CCA and whether dehydroxymethylepoxyquinomicin (DHMEQ), an effective NF-κB inhibitor, can overcome the drug resistance of CCA. Two CCA cell lines, KKU-M213 and KKU-M214, were treated with DHMEQ and/or chemotherapeutic drugs. Cell viability, apoptosis, and the expressions of the ATP-binding cassette (ABC) transporters were compared. The combination of chemotherapy drugs, 5-fluorouracil, cisplatin, and doxorubicin, with DHMEQ significantly enhanced the cytotoxicity of all chemotherapeutic drugs compared to DHMEQ or drug alone. Furthermore, the mRNA level of ABCB1, a multidrug-resistant protein, was significantly decreased in the 5-fluorouracil combined with DHMEQ-treated cells. These findings suggest that the inhibition of NF-κB by DHMEQ enhanced the chemoresponsiveness of CCA cells, possibly by reducing the expression of ABC transporter. Inhibition of NF-κB may be a potential chemodrug-sensitizing strategy for chemoresistant cancer such as CCA.

Long noncoding RNAs (lncRNAs) are known to play important roles in cancers. However, little is known about lncRNAs in cholangiocarcinoma (CCA), a cholangiocyte malignancy with poor prognosis. We investigated the role of nuclear paraspeckle assembly transcript 1 (NEAT1) lncRNA in promoting CCA. qRT-PCR analysis of patient samples showed that NEAT1 expression was higher in CCA tumors than in matched adjacent nontumor tissue. NEAT1 levels were also higher in CCA cell lines than in a normal biliary epithelium cell line (HIBEpic). NEAT1 knockdown in CCA cell lines using shNEAT1 reduced cell proliferation and colony formation in CCK-8 and colony formation assays, respectively. CCA cells transfected with shNEAT1 also exhibited reduced metastasis and invasiveness in Transwell assays. NEAT1 knockdown cells produced smaller tumors, demonstrating that NEAT1 promotes tumor growth in vivo. Silencing of NEAT1 increased E-cadherin expression in vitro, and E-cadherin expression was inversely correlated with NEAT1 expression in CCA tissue samples. RIP and ChIP assays suggest that NEAT1 is recruited to the E-cadherin promoter by EZH2 (enhancer of zeste homolog 2), where it represses E-cadherin expression. These findings indicate that NEAT1 exerts oncogenic effects in CCA. We postulate that NEAT1 is a potentially useful diagnostic and therapeutic target for CCA.

Background: Extrahepatic cholangiocarcinoma (EHCC) is the second most common primary hepatic malignancy, and is associated with high morbidity and mortality. We previously reported the decreased expression of WWOX in EHCC samples, but the underlying mechanism remained unclear. Methods: Immunoprecipitation and immunofluorescence were performed to examine the interaction of WWOX and P38 MAPK. Western blot was carried out to detect the expression of ATF2 and eIF-4E. MTT, colony formation, and Annexin V-FITC assays were performed to detect the cell proliferation and apoptosis. IHC was performed to detect the protein expression in clinical samples. Results: WWOX interacted with P38 and modulated its sub-cellular localization, leading to the cytoplasmic retention of P38. WWOX over-expression inhibited the phosphorylation of ATF2 and eIF-4E, while exogenous P38 reversed this reduction in phosphorylation. Ectopic expression of WWOX in EHCC cells led to inhibited proliferation and stimulated apoptosis in a P38 MAPK-dependent manner. In addition, we found a negative association of WWOX with nuclear localization of P38 and expression of phosphorylated ATF2 in EHCC samples. Conclusion: Our data demonstrated the role of WWOX in EHCC progression, revealing the potential of WWOX/p-ATF2 as a novel diagnostic and prognostic marker, and therapeutic target for EHCC.

Background: Cholangiocarcinoma (CCA) is highly resistant to most of the known chemotherapeutic treatments. NAD(P)H-quinone oxidoreductase 1 (NQO1) is an antioxidant/detoxifying enzyme recently recognized as an important contributor to chemoresistance in some human cancers. However, the contribution of NQO1 to chemotherapy resistance in CCA is unknown. Methods: Two CCA cell lines, KKU-100 and KKU-M214, with high and low NQO1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents; 5-fluorouracil (5-FU), doxorubicin (Doxo), and gemcitabine (Gem). NQO1 and/or p53 expression in KKU-100 cells were knocked down by siRNA. NQO1 was over-expressed in KKU-M214 cells by transfection with pCMV6-XL5-NQO1 expression vector. CCA cells with modulated NQO1 and/or p53 expression were treated with chemotherapeutic agents, and the cytotoxicity was assessed by SRB assay. The mechanism of enhanced chemosensitivity was evaluated by Western blot analysis. Results: When NQO1 was knocked down, KKU-100 cells became more susceptible to all chemotherapeutic agents. Conversely, with over-expression of NQO1 made KKU-M214 cells more resistant to chemotherapeutic agents. Western blot analysis suggested that enhanced chemosensitivity was probably due to the activation of p53-mediated cell death. Enhanced susceptibility to chemotherapeutic agents by NQO1 silencing was abolished by knockdown of p53. Conclusions: These results suggest that inhibition of NQO1 could enhance the susceptibility of CCA to an array of chemotherapeutic agents.