The present study included 68 PC patients (38 males and 30 females, 42–66 years, 52.1 ± 4.5) and 62 healthy volunteers (28 males and 24 females, 43–67 years, 52.3 ± 4.9). Inclusion criteria:1) newly diagnosed PC patients; 2) patients with no history of malignancy. Exclusion criteria:1) patients complicated with other clinical disorders; 2) patients who were treated before admission. All the participants were selected in West China Hospital, Sichuan University. Based on the staging criteria established by AJCC, there were 16, 15, 26, and 11 cases as stage I, II, III and IV, respectively. All healthy volunteers and patients signed informed consent and this study was approved by Ethics Committee of West China Hospital, Sichuan University.
Specimens and cell lines
Blood was extracted from each patient and healthy control before therapy was initiated. Blood was kept in EDTA tubes for 10 min at room temperature, followed by centrifugation at 1200g for 15 min to separate plasma.
Our study included 2 PC cell lines Capan-2 and HPAF-II to perform all in vitro cell experiments. Cells of these two cell lines were purchased from ATCC (USA). The cell culture medium was McCoy's 5a Medium Modified (10% FBS). Cell culture conditions were: 37°C and 5% CO2.
Follow-up
All patients were followed up for 5 years after admission to record their survival. Follow-up was performed every month through outpatient visit or telephone. Patients who were lost during follow-up or patient died of other diseases or accidence were not included in this study.
Real-time quantitative PCR (qRT-PCR)
RNAzol reagent (Sigma-Aldirch, USA) was used to extract total RNAs from plasma and in vitro cultivated cells. Total RNA samples were subjected to reverse transcription using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). All PCR reaction systems were prepared using SuperScript III Platinum One-Step qRT-PCR Kit (Thermo Fisher Scientific, USA) with 18S rRNA as endogenous control to analyze the expression of GAS8-AS1.
PureLink miRNA Isolation Kit (Thermo Fisher Scientific) was used to extract miRNA samples. miRNA samples were subjected to reverse transcription using TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific) was used to prepare PCR reaction systems with U6 endogenous control to analyze the expression of miR-1179.
All PCR reactions were performed in triplicate manner and data were analyzed by 2−ΔΔCT method.
Transient cell transfection
GAS8-AS1-expression vectors were constructed by Sangon (Shanghai, China). miR-1179 mimic and miRNA negative control were bought from Sigma-Aldrich (USA). All transient cell transfections were performed using lipofectamine 2000 (Invitrogen, USA) in strict accordance with manufacturer’s instructions. Subsequent experiments were performed at 24h after transfections. Two controls, including control (C, cells without transfection) and negative control (NC, cells transfected with empty vectors or miRNA negative control) were included in this experiment.
In vitro cell migration and invasion assay
Capan-2 and HPAF-II cells of different transfection groups were harvested at 24h after transfection to prepare single cell suspensions at a cell density of 3×104/ ml using serum-free McCoy's 5a Medium Modified. Cells suspensions were transferred to the upper chamber with 0.1 ml per well, while the lower chamber was filled with McCoy's 5a Medium Modified containing 20% FBS. To mimic the in vivo invading condition, upper chamber membranes were coated with 50 µg of Matrigel in culture medium at room temperature overnight. After 2 h, membranes of upper chamber were collected, cleaned and stained with 0.5% crystal violet (Sigma-Aldrich, USA) at room temperature for 20 min. Invading and migrating cells were counted under an optical microscope.
Statistical analysis
Three biological replicates were included in each experiment. Differences between PC patients and healthy controls were analyzed by unpaired t test. Differences among different cell transfection groups were analyzed by ANOVA (one-way) and Tukey test. Early diagnostic value of plasma GAS8-AS1 was analyzed using ROC curve analysis with stage I (n = 16) and stage II (n = 15) PC patients as true positive cases and healthy controls (n = 62) and true negative cases. Patients were divided in to high (n = 31) and low (n = 37) GAS8-AS1 level groups based on Youden’s index. Survival curves were plotted and compared by K-M method and log-rank t test. Correlations between plasma levels of miR-1179 and GAS8-AS1 were analyzed by linear regression. Differences were statistically significant when p < 0.05.