Cell culture, transduction and antibodies
Human cholangiocarcinoma cell lines QBC939, HCCC-9810 and HUCCT1 cells were purchased from BeNa Technology (Hangzhou, Zhejiang, China). QBC939 cells were cultured in 90% DMEM-H medium with 10% FBS containing glutamine and sodium pyruvate. HCCC-9810 and HUCCT1 cells were grow in 90% RPMI-1640 medium with 10% FBS additives containing glutamine. All culture medium was changed every 3 days and cells were humid maintained in a 37 °C 5% CO2 incubator.
QBC939 and HCCC-9810 cells (2 × 105 cells/mL) were infected with 400µL lentiviral vectors (1 × 107 TU/mL) additive with ENI.S and polybrene (10 µg/mL, Sigma-Aldrich, St Louis, MO, USA) at a MOI of 20 in 6-well plates. After cultured at 37 °C with a 5% CO2 for 72 h, the fluorescence was observed under microscope with magnification of × 100 and × 200.
Antibodies used in our study were detailed as follows: CHPF (1:100, # ab224495 Abcam) for IHC and CHPF (1:1000), E-cadherin (1:1000, #3195, CST), N-cadherin (1:1000, # ab18203, Abcam), Vimentin (1:1000, # ab92547, Abcam) for WB. Inner control antibody GAPDH (1:3000, # AP0063, Bioworld), and HRP Goat anti rabbit IgG (1:3000, #A0208, Beyotime). Ki-67 (1:200, # Ab16667, Abcam) and HRP Goat anti rabbit IgG (1:400, # ab6721, Abcam) for Ki-67 assay.
Extrahepatic and intrahepatic bile duct adenocarcinoma tissue microarray chip was obtained from Xian Alenabio Co., Ltd (Xian, Shanxi, China), including 74 cases of bile duct adenocarcinoma tissue and 5 cases of bile duct normal tissue. Before the IHC experiment, the tissue microarray chip was baked at 60 °C for 30 min in an oven. Next, the chip was dehydrated in xylene and rehydrated in graded alcohol (100%, 95%, 90%, and 70%). Antigen of the chip was recovered by boiling citric acid buffer for 30 min. After blocked with 3% H2O2 and rabbit serum, the chip was incubated with the primary polyclonal antibody of rabbit to CHPF at 4 °C overnight. Subsequently, the chip was washed with PBS three times, and the second antibody was added and incubated for 2 h at room temperature. Finally, the chip was stained with DAB solution for 10 min and counterstained with hematoxylin for 5 min. All tissues in the chip were pictured with microscopic and all slides were viewed with ImageScope and CaseViewer. IHC scores were determined by staining percentage scores and staining intensity scores. Staining percentage scores were classified as: 1 (1%-24%), 2 (25%-49%), 3 (50%-74%), 4 (75%-100%) and staining intensity were scored as 0 (Signalless color), 1 (brown), 2 (light yellow), 3 (dark brown). CHPF expression outcomes in cholangiocarcinoma tissues and para-normal tissues revealed here were analyzed.
Lentiviral vector construction and package
Short hairpin RNA of CHPF was designed in Shanghai Bioscienceres, Co., Ltd (Shanghai, China) and the sequence was 5’-AGCTGGCCATGCTACTCTTTG-3’. The following primer sequences was used for amplifying: Forward, 5’-CCTATTTCCCATGATTCCTTCATA-3’ and reverse, 5’-GTAATACGGTTATCCACGCG-3’. 20 µL PCR volume contained 0.2 µL DNA, 0.4 µL forward primer and 0.4 µL reverse primer and the PCR cycling condition is 94 °C 3 min, 94 °C 30 s, 55 °C 30 s, 72 °C 30 s, 22 cycles, a final extension for 72 °C for 5 min. The PCR products were verified by DNA sequencing and then target sequence was cloned into BR-V-108 lentiviral vector (Shanghai Bioscienceres, China). EndoFree Maxi Plasmid Kit (Tiangen Biotech, Beijing, China) was used for plasmid extraction. Qualified plasmid was used for packaging.
Transfected QBC939 and HCCC-9810 cells (LV-shCtrl and LV-shCHPF) were fully lysed and total RNA was extracted using TRIzol reagent (Sigma, St. Louis, MO, USA). Nanodrop 2000/2000C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to evaluate the RNA quality according to the manufacturer’s instructions. Promega M-MLV kit (Heidelberg, Germany) was used to reverse transcribed RNA (2.0 µg) to cDNA. Two steps qRT-PCR was performed with SYBR Green mastermixs Kit (Vazyme, Nangjing, Jiangsu, China) and melting curve was draw, the relative quantitative analysis in gene expression data were analyzed by the 2−ΔΔCt method. GAPDH act as the inner control, and the upstream and downstream primer sequences of human CHPF for the PCR reaction were 5’- GGAACGCACGTACCAGGAG-3’ and 5’-CGGGATGGTGCTGGAATACC-3’, respectively. The upstream and downstream primer sequences of GAPDH were 5’-TGACTTCAACAGCGACACCCA-3’ and 5’-CACCCTGTTGCTGTAGCCAAA-3’.
Lentivirus transfected QBC939 and HCCC-9810 cells (LV-shCtrl and LV-shCHPF) were fully lysed in ice-cold RIPA buffer (Millipore, Temecula, CA, USA) and total protein was collected. The protein concentration was detected by BCA Protein Assay Kit (HyClone-Pierce, Logan, UT, USA). A total of 20 µg per lane was separated by 10% SDS-PAGE (Invitrogen, Carlsbad, CA, USA) and then transferred onto PVDF membranes at 4 °C. The PVDF membranes were blocked with TBST solution containing 5% degreased milk at room temperature for 1 h. Then the PVDF membranes were incubated at 4 °C overnight with primary antibodies. After washing with TBST, the membranes were further incubated with second antibody horseradish peroxidase (HRP)-conjugated Goat anti rabbit IgG for 2 h at room temperature. The blots were visualized by enhanced chemiluminescence (ECL, Amersham, Chicago, IL, USA) and the density of the proteins band was analyzed by ImageJ software.
2500 lentivirus transfected QBC939 and HCCC-9810 cells were seeded into a 96-well plate with 100 mL culture medium. The detection time points were 24 h, 48 h, 72 h, 96 h, 120 h. Four hours before each detection time point, 20 µL 5 mg/mL MTT solution (GenView, El Monte, CA, USA) was added for coloring. After formazan was dissolved by DMSO solution, the absorbance values at 490 nm were measured by microplate reader (Tecan, Männedorf, Zürich, Switzerland) with a reference wavelength of 570 nm. The cell viability ratio was calculated.
Cell apoptosis and cycle assay
Lentivirus transfected QBC939 and HCCC-9810 cells were inoculated in a 6-well plate until cell density reached 85%. Cells were harvested and washed with 4 °C ice-cold PBS. After centrifugation (1200 × g), cells were resuspended with 100 µL binding buffer.
For cell apoptosis, 10 µL Annexin V-APC (eBioscience, San Diego, CA, USA) was added and incubated at room temperature without light. After 15 min later, 300 µL binding buffer was added and apoptosis analyses was measured using FACSCalibur (BD Biosciences, San Jose, CA, USA).
For cell cycle, cells were stained by staining solution containing 40 × PI (2 mg/ml), 100 × RNase (10 mg/ml) and 1 × PBS. FACSCalibur (BD Biosciences, San Jose, CA, USA) was used to detect cell cycle distribution at 200 ~ 300 Cell/s. The percentage of cells in G1, S, and G2-M phases were analyzed.
Wound healing assay
Lentivirus transfected QBC939 and HCCC-9810 cells (5 × 104 cells/well) were plated into a 96-well dish in triplicate for culturing. After confluence of cells reached 90%, the medium was exchanged to medium with 0.5% FBS. Cell scratches across the cell layer were accomplished by a 96 wounding replicator (VP scientific, San Diego, CA, USA). Than the cell layers were gently washed with PBS. 24 h and 48 h post scratching, photographs were taken by a fluorescence microscope and cell migration rates were calculated.
Human Apoptosis Antibody Array
In lentivirus transfected HCCC-9810 cells, the related genes in human apoptosis signal pathway were detected with Human Apoptosis Antibody Array (# ab134001, Abcam, Cambridge, MA, USA) following the manufacturer’s instructions. Briefly, lentivirus transfected HCCC-9810 cells at 90% confluence were collected, washed, lysed in ice-cold RIPA buffer (Millipore, Temecula, CA, USA) and then protein concentration was detected by BCA Protein Assay Kit (HyClone-Pierce, Logan, UT, USA). Protein were incubated with blocked array antibody membrane overnight at 4 °C. After washing, 1:100 Detection Antibody Cocktail was added incubating for 1 h, followed by incubated with HRP linked streptavidin conjugate for 1 h. All spots were visualized by enhanced ECL (Amersham, Chicago, IL, USA) and the signal densities were analyzed with ImageJ software (National Institute of Health, Bethesda, MD, USA).
Xenograft mouse model experiments
Female BALB/c nude mice (aged 4 weeks, weighted 17–20 g) obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were used in our study and the animal experiments were approved by Ethics committee of Hunan Provincial People’s Hospital. 4 × 106 LV-shCtrl and LV-shCHPF transfected HUCCT1 cells were subcutaneously injected into the 10 mice which were randomly divided into shCtrl and shCHPF groups. The mice were housed in a conditional environment with a 12-h dark/light cycle. The tumor size and weight of each mouse was monitored 2 times per week. For in vivo bioluminescence imaging, all mice were anesthetized by intraperitoneal injection of 0.7% pentobarbital sodium (10 uL/g) 9 weeks post cell injection, and anesthetized mice were placed under the Berthold Technologies living imaging system and imaging was collected. Then anesthetized mice were sacrificed by cervical dislocation and the tumor tissues were harvested for Ki-67 staining assay.
Ki-67 staining assay
Mice tumor tissues were fixed in 10% formalin for 24 h and then were paraffin-embedded. 5 µm slides were cut and immersed in xylene and ethanol for deparaffinization and rehydration. Tissue slides were blocked with 3% PBS-H2O2 and were incubated with primary antibody Ki-67 at 4 °C overnight. Then slides were incubated with HRP goat anti-rabbit IgG at room temperature for 2 h. Finally, all slides were stained by Hematoxylin (# BA4041, Baso) for 10 min and Eosin for 5 min (# BA4022, Baso, Zhuhai, Guangdong, China). Stained slides were examined at × 100 and × 200 objective lens microscopic.
All cell experiments were performed in triplicate and data were shown as mean ± SD. The significance of the differences between shCHPF and shCtrl group in cells experiment was determined using the two-tailed Student’s t-test. Mann-Whitney U analysis and Spearman rank correlation analysis were used while explaining the relationship between CHPF expression and tumor characteristics in patients with cholangiocarcinoma. Statistical significance (P value) was calculated by SPSS 22.0 (IBM, SPSS, Chicago, IL, USA) with P value < 0.05 as statistically significant. Graphs were made using GraphPad Prism 6.01 (Graphpad Software, La Jolla, CA, USA).