Patients and tissue samples
Human CCA tissues and matched normal tissues were obtained from 42 CCA patients who received surgery at the First Affiliated Hospital of Jinzhou Medical University (Jinzhou, China). No patients underwent chemotherapy or radiotherapy before surgery. Tissue samples were collected and used with written informed consent from all patients. This study was approved by the Ethics Committee of Jinzhou Medical University.
Cell lines and cell culture
Human CCA cell lines (HUCCT1 and QBC939) and the normal bile duct epithelial cell line HiBEC were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in RPMI-1640 medium (Gibco, Rockville, MD, USA) containing 10% fetal bovine serum (FBS, Gibco) and 100 μg/mL penicillin/streptomycin and incubated in a humidified atmosphere with 5% CO2 at 37°C.
Quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and synthesized into cDNA using PrimeScript RT reagent kit (Takara, Japan). RT-qPCR was performed with SYBR Premix Ex Taq™ (Takara, Japan) on the ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The following primers were used: TRIM66, 5’-GCCCTCTGTGCTACTTACTC-3’ (forward) and 5’-GCTGGTTGTGGGTTACTCTC-3’ (reverse), GAPDH, 5’-CCATCAATGACCCCTTCATTG-3’ (forward) and 5’-CATGGGTGGAATCATATTGGAAC-3’ (reverse). The relative expression of target genes was normalized to GAPDH. All data were calculated using the 2−ΔΔCt method.
Western blot analysis
Tissues or cells were lysed in lysis buffer for protein extraction. Protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). An equal amount of protein was separated by 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk, followed by overnight incubation at 4°C with primary antibodies against TRIM66, E-cadherin, α-catenin, N-cadherin, vimentin, p-PI3K, PI3K, p-Akt, Akt and GAPDH. After washing with TBST, the membranes were incubated with appropriate secondary antibody. All antibodies in this study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein bands were detected using an ECL kit (Pierce, Rockford, IL, USA) and their intensity was quantified using the ImageJ software.
Cell transfection
TRIM66 shRNA (shTRIM66) and the control shRNA (shNC) were designed and synthesized by GeneCopoeia (Rockville, MD, USA). Cells were cultured in a 96-well plate at a density of 1×103 cells/well. After reaching 80% confluence, cells were transfected with shTRIM66 or shNC using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The western blot analysis was performed to confirm the transfection efficiency.
MTT assay
Cell proliferation was assessed using the MTT assay. In brief, cells were seeded in a 96-well plate at a density of 1×103 cells/well. After incubation for different time, MTT (5 mg/mL, Sigma, St. Louis, MO, USA) was added to each well and cells were cultured for another 4 h. Subsequently, the supernatants were removed from each well and DMSO (Sigma) was added. The absorbance value was measured at 570 nm using a microplate reader.
Wound healing assay
Cells were seeded in a 6-well plate and cultured to reach 80% confluence. A sterile plastic tip was used to scratch a wound on cell monolayer and then cells were cultured for 24 h. The images of wounds were taken at the indicated time points under a microscope (200×).
Transwell assay
Transwell chambers were used to measure cell invasion. Cells were resuspended in serum-free medium and added to the upper chamber. The membrane was coated with Matrigel. The lower chamber was filled with 10% FBS as chemoattractant. After incubation for 24 h, cells remaining on the upper surface of the membrane were removed and invading cells on the lower surface of the membrane were fixed and stained with crystal violet. The number of invading cells in five random fields was counted under a microscope (200×).
Measurement of glucose uptake and lactate production
48 h after transfection, cell culture media was collected to determine glucose uptake and lactate production using a glucose assay kit (Sigma) and a lactate assay kit (Sigma), respectively. Glucose and lactate levels were calculated with normalization to corresponding protein levels.
Statistical analysis
Data were collected from three independent experiments and shown as means ± standard deviation (SD). The SPSS software version 19.0 was used for statistical analysis. Student’s t-test or one-way ANOVA was applied for the comparison between different groups. P<0.05 was considered statistically significant.