Study subjects of the present study were 60 GBC patients (gender: 26 males and 34 females; age: 58 to 79 years, 69.3±6.6 years). Those patients were selected from the 133 GBC patients admitted to Hubei Provincial Cancer Hospital between May 2015 and January 2018. Inclusion criteria: 1) all cases were newly diagnoses GBC cases; 2) therapies were not initiated. Exclusion criteria: 1) patients transferred from other hospital; 2) recurrent cases; 3) patients complicated with other severe clinical disorders. According to AJCC staging methods and clinical findings, there were 12, 13, 18 and 17 cases at stage I-IV, respectively. This study passed the review board of aforementioned hospital Ethics Committee (2015HBCH57493749). All patients were informed with experimental details and informed consent was signed.
GBC specimens and cells
Before any therapies, paracancerous and GBC tissues were collected from all the 60 GBC patients through biopsy. All tissues were subjected to pathological examinations and all tissues were correct.
Fasting blood (3ml) was also extract from all GBC patients before any therapies. Blood was added into EDTA-treated tubes. Following centrifugation at 1200 xg for 15min, supernatant (plasma) was collected.
GBC-SD and SGC-996 two human GBC cell lines (Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultivated in DMEM cell culture medium (10% FBS). Cells culture conditions were 5% CO2, 37 °C and 95% humidity.
Vectors and transient transfections
EPIC1 and LET expression vectors were constructed using pcDNA3 vector (RIBOBIO, Guangzhou, China). GBC-SD and SGC-996 were harvested and counted. Lipofectamine 2000 (Invitrogen, USA) was used to achieve transient transfections of 10 nM vectors (empty pcDNA3 vector as a negative control, NC) into 106 cells. Control (C) cells were cells without transfections. The interval between transfections and following experiments was 24h.
Total RNA extractions
Tissues were ground in liquid nitrogen. Total RNAs in 0.01g tissue, 0.2 ml plasma and 106 cells were extracted using Trizol reagent (Invitrogen, USA). RNA samples were precipitated and washing using 75% Ethanol. All RNA samples were stored in liquid nitrogen before following experiments.
Total RNA samples were digested with DNase I for 1h at 37 °C to remove genomic DNAs. The digested RNA samples were used as template to perform reverse transcriptions using AMV reverse transcriptase (GIBCO, USA). With GAPDH as endogenous control, qPCR reaction mixtures were prepared using QuantiTect SYBR Green PCR Kits (Qiagen) to detect the expression of EPIC1 and LET. Three replicate tubes were set for each reaction and all Ct value normalizations were performed using 2-ΔΔCq method.
Cell proliferation analysis
EPIC1 and LET were harvested. Cells were counted and 1ml DMEM cell culture medium (10% FBS) was mixed with 3×104 cells to make single cell suspensions. In an 96-well cell culture plate (0.1 ml per well), cells were cultured under conditions of 5% CO2, 37 °C and 95% humidity. Three replicate wells were set for each transfection group. Following that, 10ul CCK-8 solution (Sigma-Aldrich) was added into each well at 2h before the termination of cell culture. After cell culture was ended, 10ul DMSO was added into each well and OD values were measured at 450 nm.
Cell apoptosis analysis
EPIC1 and LET were harvested. Cells were counted and 1ml DMEM cell culture medium (0.1% FBS) was mixed with 3×104 cells to make single cell suspensions. In a 6-well plate (2ml per well), cells were cultured under conditions of 5% CO2, 37 °C and 95% humidity. Three replicate wells were set for each transfection group. Cell culture was performed for 48h, followed by cell digestion using 0.25% trypsin. Following staining using propidium iodide (PI) and Annexin V-FITC (Dojindo, Japan), flow cytometry was performed to separate apoptotic cells.
All qPCR, cell proliferation and cell apoptosis experiments were repeated 3 times. Data were expressed as mean values. Differences were explored by either paired t test (GBC vs. paracancerous) or ANOVA (one-way) combined with Tukey test (among different cell transfection groups). Correlations were analyzed by linear regression. p<0.05 was statistically significant.