Plant materials and extraction
Crowberry was provided by the Key Laboratory of Natural Medicines of the Changbai Mountain (Yanbian University), Ministry of Education. The whole plant of crowberry was purchased in March 2018 at the Gen He Crude drug market in Hei Long Jiang Province, China. The voucher specimen (No. YBU-1020) was identified by Professor Hui-zi Lv, Botanical garden, Yanbian University, and stored in the Herbarium of the College of Pharmacy at Yanbian University (China). The air-dried and powdered whole plant of crowberry (6.0 kg) were extracted with MeOH (25 L, reflux, 5 h × 3), followed by removal of the solvent under reduced pressure to yield a dried MeOH extract (1.5 kg, yield 25%). The dried MeOH extract was dissolved in dimethyl sulfoxide (DMSO) to a density of 300 mg/ml and deposited at − 20 °C until analysis.
High-performance liquid chromatography and HPLC–tandem mass spectrometry (MS/MS)
Chromatographic experiments were performed using an HPLC instrument equipped with a 5260 autosampler and 5430 diode-assay UV/Vis detector (DAD) (HITACHI Chromaster, Japan) and mass spectrometry (Agilent, 6460, USA). For all experiments, a LaChrom ODS C-18 column (250 mmL × 4.6 mmI.D., 5 μm, HITACHI, Japan) was used as the stationary phase, and the syringe amount was 5 μL. The mobile phase was composed of 0.5% phosphoric acid solution (A) and methanol (D), and the system was equilibrated for 20 min with the beginning conditions. The following gradient program has been applied to methanol (D): linear gradient from 58% D to 60% D for 20 min, 60% D increasing to 62% D in 55 min, and then 62% D increasing to 100% D for 20 min. The column was washed with 100% D for 10 min, and the flow rate was 0.7 mL/min. UV absorption at 250 nm was measured to define the analyses (Fig. 1). Then obtain the corresponding chromatographic peaks for 2'-methoxy-4'-hydroxy-dihydrochalcone(1), 2',4'-dihydroxy-dihydrochalcone(2), and 2',4'-dihydroxy-chalcone (3).
Cell lines and cell culture
CCA cell lines, RBE and QBC939, were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The human cholangiocarcinoma cell lines, HuCCT1 and TFK1, were gifted by Kanazawa University in Japan. Normal human bile duct epithelial cell HIBEpic was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines above were grown in RPMI-1640 medium (GIBCO, Gaithersburg, MD). Cells were supplemented 1% penicillin/streptomycin and 10% fetal bovine serum (GIBCO, Gaithersburg, MD). Cells were incubated at 37 °C with a 5% CO2 atmosphere.
MTT assay
HuCCT1 and QBC939 cells were seeded 5000 cells/well in a 96-well plate, and treated with crowberry at various concentrations for 24, 48, 72 h. Correspondingly, silenced and overexpressed DEK cells were cultured for 48 h. Briefly, the value of absorbance was measured at a full-wavelength spectrophtotmeter (TECAN-infinite M200 pro, Switzerland).
Colony formation assay
Cells were seeded in six-well plates and then cultured with crowberry for 2 weeks. The visible colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet (Solarbio, China). ImageJ software was used to analysze the data.
EdU assay
Cells were seeded at 5000 cells/well in 96-well plates. After treatment, these cells were cultured in complete medium containing 50 µM EdU (RiboBio, China) for 2 h at 37 °C followed by washing twice with phosphate-buffered saline (PBS). The cells were fixed and permeabilized and then, EdU was detected following the EdU assay kit manufacturer’s instructions. Finally, cell immunostaining was observed and images were acquired with a fluorescence microscope (Olympus, Japan).
Hoechst33342 staining
After treating with the indicated crowberry for 48 h, cells were fixed with 4% formaldehydefor, and stained for 15 min with 1 μg/ml Hoechst33342 in the dark. The cells were then washed twice with PBS. A fluorescence microscope was used to image the cells.
Flow cytometry
Cells treated with different concentrations of crowberry after 48 h. The cells were washed with PBS and re-suspended in binding buffer. Cells were stained in the dark with FITC-annexin V and PI (BD Biosciences, USA) for 15 min. BD Accuri C6 (BD Biosciences) was used to detect the samples.
Wound healing assay
Cells were cultured in 6-well plate to 80% confluence. The wounds were created by 200 µl micropipette tip and replaced with different doses of crowberry. Cell migration was observed at 0, 12 and 48 h using microscopy (Olympus, Japan). Silenced or overexpressed DEK cells were observed at 12 h. Image J software was used to analyze the results.
Cell migration assay
Cells were seeded in the upper chambers of Transwell inserts (Costar, Corning Incorporated, USA), at a concentration of 5 × 104 cells/well. Then the cells were incubated with medium containing 1% fetal bovine serum (FBS) for 4 h. Medium containing 10% or 20% FBS was added to the lower chamber. Specifically, QBC939 cells were cultured for 72 h, and HuCCT1 cells were cultured for 24 h. Cells were fixed with 4% paraformaldehyde and then stained with 1% crystal violet. Cells were photographed with an Olympus BX53 microscope.
Transfection
DEK small interfering RNA (siRNA) reagent was purchased from RiboBio (China). The effective sequence of the DEK gene used here was (5'-TGTCCTCATTAAAGAAGAA-3). The cells were incubated with 50 nM siRNAs containing Lipofectamine 3000 (Invitrogen, Carlsbad, CA) for 48 h. An lentiviral vector overexpressing DEK (DEK (Human, NM_003472)) was purchased from Beijing Syngentech Co., Ltd, China, which serial number is pHS-AVC-0466. HuCCT1 cells were transfected with this vectorin a 6-well plate, and 2 ml of medium supplemented with 2 µl of polybrene and 2 µl of lentivirus was added for transfection. After 8 h on incubation, the medium was replaced with fresh medium, and the cells were incubated for another 72 h. The DEK-transduced cells were incubated with 5 μg/ml puromycin in the medium.
Western blotting
After the cells were treated with crowberry for 48 h, total proteins (40 μg) were quantified with BCA protein assay (Beyotime, Shanghai, China), electrophoresed through SDS-PAGE, and transferred to PVDF membrane (Millipore, Eschbom, Germany). The membrane was incubated with primary antibodies Bax (1:1000), Bcl-2 (1:1000), Cleaved-Caspase3 (1:1000), Cleaved-Caspase9 (1:1000), Cleaved-PARP (1:1000), Cytoc (1:1000), p-Akt (1:1000), Akt (1:1000), p-S6 (1:1000), S6 (1:1000), p-4EBP1 (1:1000), 4EBP1 (1:1000), E-cadherin (1:1000), Vimentin (1:1000), and Snail (1:1000), which were obtained from Cell Signaling Technology, USA, and DEK (1:1000, BD, USA), actin (1:3000, Abcam, USA) at 4 ℃ overnight. Secondary antibody (1:3000, Beyotime, China) was added at RT for 1 h. Enzymatic signals were visualized with a ChemiDoc Touch Imaging System (Bio-Rad, USA), and statistics were performed with ImageJ software.
Xenograft model
The study was approved by the Ethics Committee of Yanbian University. BALB/C female nude mice (4 weeks old) were obtained from Changzhou Cavens Laboratory Animal Co., Ltd., China. Each mouse was injected with 2 × 106/100 µl QBC939 cells subcutaneously into the rigtht armpit. One week after inoculation, 16 mice were randomly assigned into 2 groups (8 mice in each group). Every day, mouse body weight was recorded, and tumors were measured with a Vernier caliper; the tumor volume was ascertained following the formula: tumor volume = 0.5 × (length × width2). The treatment group was treated every day with crowberry (50 mg/kg) diluted with 0.1% methylcellulose for intragastric administration (ig), and the same volume of methylcellulose was used to treat the control group mice. On day 30, the mice were anesthetized with 1% pentobarbital sodium and then sacrificed. Mouse livers and tumors were fixed in 4% paraformaldehyde for immunohistochemical and hematoxylin and eosin (H&E). This experiment was completed in a specific-pathogen-free (SPF) animal laboratory in the Yanbian University Animal Experiment Center. The experiment was approved by the Animal Protection Committee of Yanbian University (SCXK (JI)2017–0003). the research was conducted in accordance with internationally accepted principles (European community guidelines) for laboratory animal use and care.
Immunohistochemistry staining (IHC)
The fixed mouse tumors were embedded in paraffin and then sliced into 4 µm thin slices. Antigen was retrieved in 100 °C citrate buffer for 10 min. After endogenous peroxidase was blocked with 1% hydrogen peroxide for 10 min, the Ki67 antibody (1:100, Affinity Biosciences, rabbit monoclonal, USA) and DEK antibody (1:100, protheintech, Mouse monoclona, USA) were incubated at 4 ℃ overnight. After incubation with secondary antibody, the section was observed with diaminobenzidine (DAB, Zhongshan Golden Bridge PV- 9000, Beijing, China), and the nucleus was stained with hematoxylin. Five fields of 400 × view were selected to count the number of Ki67-positive cells.
Statistical analysis
Statistical analyses were primarily conducted by GraphPad Prism 8.0 software (GraphPad, San Diego, CA) and SPSS 20.0 software (SPSS, Chicago, IL). One-way ANOVA was used for analysis multiple comparisons. Two group comparisons were performed using T-test. P-Values < 0.05 were considered as significant.