Human tissues were provided by the Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University. Paired tumor and adjacent non-tumor tissues were obtained from 5 patients with CCA (1 male and 4 females with 53-70 years old) who underwent surgery at Department of Surgery at Srinagarind Hospital, Faculty of Medicine, Khon Kaen University. Written informed consent was obtained from subjects (HE521209). Experimental design and procedures were approved by Naresuan University Institutional Review Board for Human Ethic (approval no. NU-IRB0895/61). For the CCA samples, five tissues were selected from clearly differentiated tumor and non-tumor areas, as selected from the tissues as in a previous study . These tissues included paraffin-embedded tissues of patients with intrahepatic CCA who underwent liver resection. Diagnosis was evaluated using clinical data, imaging analysis, tumor markers and pathology. Immunohistochemical studies for pathological diagnosis included antibodies against cytokeratin-7 (CK7), cancer antigen or carbohydrate antigen 19-9 (CA19-9), hepatocyte paraffin 1 (HepPar1) and α-fetoprotein. The tumor tissues were verified as CCA based on the following criteria when either CK7+ or HepPar1-, with or without CA19-9+, were found. The study protocol (previous sample collection) was approved by The Human Research Ethics Committee, Khon Kaen University, Thailand (approval no. HE551407) before the study was conducted.
Establishment of stable AGR2vH-overexpressing cells
The procedure of AGR2vH-overexpressing cell establishment was conducted as described in our previous studies [13, 14]. The AGR2vH amplicon was amplified, sequenced and digested with HindIII and EcoRI restriction enzymes (Thermo Fisher Scientific, Inc.), before being inserted into p3XFLAG-CMV-14 expression vector (Sigma-Aldrich; Merck KGaA). The pCMV14-AGR2vH and pCMV14-empty vectors were transfected into KKU-213A cells using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) and the single clone was selected using Geneticin G418 (Thermo Fisher Scientific, Inc.). The successful establishment of AGR2vH overexpressing cells was confirmed using reverse transcription (RT)-PCR and RT-quantitative (q)PCR.
Cell line and cell culture
The human CCA cell lines, KKU-213A  and KKU-213L5 , were provided by the Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% v/v FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2.
RNA extraction, RT-PCR and RT-qPCR
Total RNA was extracted from cells or tissues using RiboZol™ reagent (Amresco LLC) following the manufacturer’s instructions. cDNA synthesis was performed using a HiSenScript™ RH(-) cDNA Synthesis kit (Intron Biotechnology, Inc.).
The PCR reaction was performed using 1X MyTaqTM HS Red mix (Bioline; Meridian Bioscience) under optimized conditions using AGR2vH specific primers, which are described in previous studies [13, 14]. β-actin was used as an internal control for semi-quantitative normalization. The amplification procedure consisted of the following steps: Initial denaturation at 5 min at 94˚C, followed by 28 cycles of denaturation for 30 sec at 94˚C, annealing for 30 sec at the primers specific annealing temperature, extension for 30 sec at 72˚C, and a final extension for 7 min at 72˚C. PCR products were analyzed via 1% agarose gel electrophoresis, detected using ImageQuant™ LAS 500 (Cytiva) and quantitated using ImageQuant TL 7.0 software (GE Healthcare). RT-qPCR was performed for relative quantification of gene expression using 1X LightCycler® 480 SYBR Green I Master (Roche Applied Science) under optimized conditions (13,14), and analysis was conducted using the LightCycler® 480 system (Roche Applied Science). The expression levels of the target genes were normalized with β-actin using the relative quantification formula of 2-ΔΔCq .
Cell proliferation assay
Cell proliferation was examined using a Cell Counting Kit-8 (CCK-8) kit (Dojindo Molecular Technologies, Inc.) using water soluble tetrazolium substrate. Cells were plated at 3,000 cells per well in a 96-well plate. After incubation for 24, 48 and 72 h, 10 µl CCK-8 reagent was added in to each well and incubated at 37˚C for 2 h, and the absorbance was measured at 450 nm. The average absorbance at 450 nm of the determinate cells at 24 h in each group was normalized as 1.00 for calculation of relative of cell proliferation. The experiment was performed independently in biological triplicate.
Colony formation assays
Anchorage-dependent growth was evaluated using a clonogenic assay in 6-well plates. pCMV14-empty and pCMV14-AGR2vH cells were seeded at 500 cells per well and incubated at 37˚C in a humidified atmosphere of 5% CO2 for 14 days. Colonies were fixed in 4% paraformaldehyde, stained with crystal violet solution and counted under a light microscope with 4X magnification.
All animal studies adhered to protocols approved by the Naresuan University Institutional Animal Care and Use Committee (Project no. NU-AE610623; Approval no. 6101025). Twelve Male BALB/CAJcl-Nu/Nu mice (age, 4-6 weeks) were obtained from Nomura Siam International Co., Ltd., housed under specific pathogen-free conditions and cared for according to the institutional guidelines for animal care.
In total, six mice were used in each group to establish the subcutaneous xenograft model. Then, 2.5x106 pCMV14-AGR2vH4 transfected KKU-213A cells or pCMV14-Empty transfected KKU-213A cells in 150 μl PBS were subcutaneously injected into the right flanks of nude mice. The body weight of the mice and the diameters of tumors were monitored every 2 days for 3 weeks, and tumor volume (mm3) was calculated using the following equation: Tumor length (mm) x tumor width (mm)2/2. All animals were sacrificed on day 21 after xenograft transplantation using an overdose of sodium pentobarbital (≥100 mg/kg) via intraperitoneal injection. Tumors were excised and tumor weight was measured.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and protein bioinformatics
Total protein was extracted from pCMV14-AGR2vH4 transfected KKU-213A cells or pCMV14-Empty transfected KKU-213A cells, using RIPA buffer (0.05 M Tris-HCl pH 7.4, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.15 M sodium chloride, 0.002 M EDTA and 0.05 M sodium fluoride) and measured using a Bradford assay. The concentration of each protein sample was adjusted to 10 μg/μl and subjected to a phosphoprotein enrichment procedure (Pierce; Thermo Fisher Scientific, Inc.). Then, 50 μg protein was subjected to in-solution digestion, mixed with 50 ng/µl trypsin (Promega Corporation) and incubated at 37˚C overnight. The digested samples were dried and protonated with 0.1% formic acid before injection into LC-MS/MS. Tryptic peptide samples were prepared for injection into an Ultimate3000 Nano/Capillary LC system (Thermo Fisher Scientific, Inc.) coupled to a Hybrid quadrupole Q-Tof impact II™ (Bruker Daltonics) equipped with a Nano-captive spray ion source. Electrospray ionization was carried out using CaptiveSpray. Mass spectra were obtained in the positive-ion mode over the range (m/z) 150-2,200 (Compass 1.9 software; Bruker Daltonics). MaxQuant 126.96.36.199 was used to quantify the proteins using an Andromeda search engine to correlate MS/MS spectra to the Uniprot Homo sapiens database .
Only peptides with a minimum of seven amino acids and ≥1 unique peptide were required for protein identification. Only proteins with ≥2 peptides and ≥1 unique peptide were considered as being identified and used for further analysis. As a search FASTA file, 954 proteins present in the Homo sapiens proteome downloaded from Uniprot. The proteome data obtained were further analyzed via UniProt, Gene Ontology (GO), Vocalno plot, Heat map and Panther analyses.
Protein extraction and western blotting
For whole cell protein lysate, cells were lysed with RIPA lysis buffer containing 7 M urea, 2 M thiourea, 4% 3‑[(3‑cholamidopropyl) dimethylammonio]‑1‑propanesulfonate and protease and phosphatase inhibitors. For cytoplasmic and nuclear protein preparation, cells were washed with cold PBS, and harvested in cell lysis buffer (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl and 0.5 mM DTT), and the supernatants were first collected as the cytoplasmic fraction. Then, the nuclear pellets were collected, washed twice with ice cold PBS and lysed with nuclear lysis buffer (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and 0.5 mM DTT) . Phosphatase inhibitors and protease inhibitors (Roche Diagnostics GmbH) were added at each step.
The protein concentration was determined using the Bradford assay. The 40, 20 and 10 µg whole cell proteins, cytoplasmic protein and nuclear protein, respectively, were separated via 12% SDS-PAGE and transferred to a PVDF membrane (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk, the membrane was incubated with primary antibodies overnight at 4˚C. The primary antibodies were used at a dilution of 1:500 for b-catenin (Elabscience, Inc.), AKT (Elabscience, Inc.) and phosphorylated (p)-AKT (Elabscience, Inc.), and 1:5,000 for GAPDH (Sigma‑Aldrich; Merck KGaA). After incubation with specific secondary antibodies (1:2,000), the expression of protein was observed using an ECL solution (Bio-Rad Laboratories, Inc.).
Experiments were performed in triplicate. Data are presented as the mean ± SD. An unpaired Student’s t-test (two tailed) was used for comparison between each group using SigmaPlot software (SigmaPlot 11.0; Systat Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.