YAP is a critical oncogene in human cholangiocarcinoma.

Yes-associated protein (YAP), a transcriptional co-activator, has important regulatory roles in cell signaling and is dysregulated in a number of cancers. However, the role of YAP in cholangiocarcinoma (CCA) progression remains unclear. Here, we demonstrated that YAP was overexpressed in CCA cells and human specimens. High levels of nuclear YAP (nYAP) correlated with histological differentiation, TNM stage, metastasis and poor prognosis in CCA. Silencing YAP increased tumor sensitivity to chemotherapy and inhibited CCA tumorigenesis and metastasis both in vivo and in vitro. YAP overexpression in vivo and in vitro promoted CCA tumorigenesis and metastasis. Additionally, we found that YAP induced epithelial-mesenchymal transition (EMT) and formed a regulatory circuit with miR-29c, IGF1, AKT and gankyrin to promote the progression of CCA. Results of CCA tissue microarray showed positive correlations between nYAP and gankyrin or p-AKT expression. Combination of nYAP and gankyrin or p-AKT exhibited improved prognostic accuracy for CCA patients. In conclusion, YAP promotes carcinogenesis and metastasis by up-regulating gankyrin through activation of the AKT pathway.


RNA-interference
The CCA cells were transfected with siRNAs by using Lipofectamine

Western blot
Procedure for western blot was performed routinely, as reported previousl

Coimmunoprecipitation (co-IP) assay
The cells were lysed in 400 μl lysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaC1, 10 mM EDTA and 0.5% NP-40) containing protease inhibitors. The lysates were incubated with 15μl of anti-Gankyrin monoclonal antibody at 4°C for 2 h. The complex were then precipitated with 15μl protein A-Sepharose and incubated for 4h at 4°C with gentle rotation. After washing with lysis buffer, the beads were boiled with loading buffer and analyzed by Western blotting. Normal mouse IgG was used as a negative control.

Taqman Real-Time PCR Analysis
TaqMan real-time PCR was performed as described using commercially available primers designed against human YAP, Gankyrin and GAPDH [1]. Total RNA was extracted from cells and tissues using Trizol

Cell cycle analysis
Cells (4x10 5 ) were fixed in 70% ethanol for 1 h at 4˚C. Then the cells were washed twice with PBS and 10 mg/mL RNase A was added. After that propidium iodide was added to the tubes at a final concentration of 0.05 mg/ml, and the samples were incubated at 4˚C for 30 min in a dark environment. The result was analyzed by flow cytometry (Beckman Coulter EPICSALTRA II).

Apoptosis assay
CCA cells (2 x10 5 /well) were cultured in six-well plates, and then were collected and washed twice with ice-cold PBS. Apoptosis was investigated by flow cytometry using Annexin V-PE Apoptosis Kit (Becton Dickinson, San Diego, CA) following the protocol.

Wound-Healing Assay
For the wound-healing assay, CCA cells were cultured in six-well plates until confluence and then scratched with a 10μL pipette tip. Then images were captured at 0 and 24h hours after scratching.

Migration and Invasion Assay
Cell motility and invasive capacites were investigated by way of transwell (BD Biosciences, San Jose, CA, USA) and Matrigel invasion (BD Biosciences), respectively. For transwell migration assay, 2-3x10 4 cells were seeded, whereas 3-4x10 4 cells were seeded for the invasion assay. The assay had been performed for 36 h at 37 °C, non-migrated or non-invaded cells were removed from the upper surface of the membrane.
Cells migrated to the underside of the membrane were fixed and stained with 0.5% crystal violet, and then were counted under an optical microscope. Each experiment was repeated at least three times.

Chromatin immunoprecipitation assay
CCA cells were cross-linked by the addition of formaldehyde to a 1% final concentration, the chromatin was sonicated, and the immunoprecipitation was performed using 1 µg of YAP and IgG antibody.
ChIP assays were performed using a commercially available ChIP assay kit (Simple ChIP Cell Signaling Technology) following the manufacturer's instructions. The sequence of primers for gankyrin used to amplify ChIP enriched DNA is available upon request. The mRNA level of gankyrin were evaluated by RT-PCR.

Construction of tissue microarrays and immunohistochemistry
CCA samples and specimens of nonneoplastic tissues were used to construct a tissue microarray (Shanghai Biochip Co., Ltd. Shanghai, China). Expression of YAP, gankyrin, p-AKT, Ki-67, E-cadherin, and N-cadherin in tumor tissues was detected by IHC analysis as described previously [1]. The density was evaluated by Image-Pro Plus v6.2 software (Media Cybernetics Inc, Bethesda, MD). For the reading of each antibody staining, a uniform setting for all the slides was applied.
Integrated optical density of all the positive staining in each photograph was measured, and its ratio to total area of each photograph was calculated as density. For YAP density, the cutoff for the definition of subgroups was the median value. Samples were then segregated into two groups for each analysis. The first group comprised samples in which YAP expression was above the median value (YAP-high group), and the second group comprised the rest of the samples (YAP-low group). Each data set was analyzed separately and consensus evaluation from at least two of the three investigators was considered acceptable.