Cell lines and culture conditions
Human PC cell lines (SW1990 and PANC-1), obtained from the American Type Culture Collection (ATCC; Rockville, USA), were cultured as described previously [18]. GR PC cells (PANC-1/GR and SW1990/GR) were selected from their respective parental cells that were exposed to gradually increasing concentrations of gemcitabine (Sigma-Aldrich, St. Louis, USA) for more than 6 months. These cells were then maintained in high-glucose Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Waltham, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, USA) in a humidified atmosphere containing 5% carbon dioxide at 37˚C.
MTT assay
A total of 5,000 cells were plated in 96-well plates for overnight incubation and then treated with different concentrations of gemcitabine for 48 h. According to the manufacturer's instructions, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) assay was performed to determine the cell viability. SPSS statistical software (version 24.0; Chicago, USA) was used to calculate the concentration of gemcitabine required for 50% growth inhibition (IC50). All assays were performed thrice independently.
Transwell assay (cell migration and invasion)
The migration and invasion capacities of the cells were detected using the Transwell assay, performed according to the manufacturer's instructions, as described previously [19]. Briefly, in the invasion assay, 5 × 104 cells were added to the transwell chambers precoated with MatrigelTM (BD Biosciences, San Jose, CA, USA). Then, the chambers were placed in a 24-well plate (Corning, New York, USA) and incubated for 24 h. After fixing with 4% paraformaldehyde and staining with 0.1% crystal violet, the number of invading cells was counted using a microscope (Olympus, Tokyo, Japan) in five randomly chosen fields. The same steps were performed in the cell migration assay, except that MatrigelTM was not used.
RNA extraction and quantitative RT-PCR assay
Total RNA was isolated from the cell lines using Trizol reagent (Invitrogen, Thermo Fisher Scientific, Inc.), then reverse transcribed using the PrimeScript RT reagent kit or the PrimeScript miRNA cDNA Synthesis Kit (TaKaRa, Dalian, China), according to the manufacturer's instructions. Then, qPCR was performed using SYBR Premix Ex Taq II kit (Takara) or Taqman miRNA kit (Applied Biosystems, Foster City, CA), following the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were regarded as the internal controls, and the relative gene expression was determined using the 2−ΔΔCt method. The primer sequences used are listed in Table 1.
Western blotting
The cells were lysed using radioimmunoprecipitation assay buffer (RIPA buffer) (Beyotime, Guangzhou, China) containing protease inhibitors. Protein concentrations were detected using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Protein samples were separated using 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and then incubated with primary antibodies. The antibodies against E-cadherin (#14472, 1:1000), N-cadherin (#13116, 1:1000), vimentin (#5741, 1:1000), Snail (#3879, 1:1000), ZEB1 (#3396, 1:1000), and ZEB2 (#97885, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA) and the anti-GAPDH antibody (ab8245, 1:2000) was obtained from Abcam (Cambridge, MA, USA). All the antibodies were used according to the manufacturers’ instructions.
Transfection ofNEAT1 short hairpin RNA
Lentivirus (GV118)-encoding short hairpin RNA (shRNA) targeting NEAT1 and its negative control oligonucleotides were synthesized by Shanghai GenePharma Co. Ltd (Shanghai, China). Hsa-miR-506-3p mimics and inhibitors along with their corresponding negative controls were prepared by Research-Bio Co., Ltd (Shanghai, China). Cells were transfected using Lipofectamine 2000 (Invitrogen) when PANC-1/GR and SW1990/GR cells at the logarithmic growth phase. The transfection efficiency was confirmed using qRT-PCR. In the specified group, cells were exposed to 10 ng/mL TGF-β (R&D Systems, Minneapolis, USA) 24 h after transfection and then allowed to incubate at 37°C for 48 h. For generation of stably transfected cells, the cells were treated with 2 μg/mL puromycin (Sigma-Aldrich) for two weeks, following which the GFP-positive cells were selected for subsequent assays.
Cell apoptosis assay
Cell apoptosis assay was performed using the Annexin V/PI staining kit (Sungene Biotech, Tianjing, China), as described previously [19]. In brief, cells (1×105) were harvested for each assay, washed with cold PBS, and resuspended in 200 mL binding buffer. Then incubated with Annexin V-fluorescein isothiocyanate (5 µl) for 10 min and propidium iodide (PI) for 5 min at room temperature in the dark. Finally, the samples were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA) within 1 h.
Dual-luciferase reporter assay
The online software StarBase3.0 (http://starbase.sysu.edu.cn/) was used to predict the binding sites of NEAT1 to mi-506-3p. ZEB2 was a potential target of miR-506-3p by bioinformatical prediction tools, TargetScan 7.2 (http://www.targetscan.org/vert_72/). The fragment from NEAT1 or 3'UTR of ZEB2 containing the predicted binding sites of miR-506-3p were amplified using PCR, and cloned into the XhoI and XbaI sites of the downstream of Firefly luciferase gene in the pmirGLO vector (Promega, Madison, USA) to generate luciferase reporter vectors NEAT1-wt and ZEB2-3'UTR-wt, respectively. To test the binding specificity, the corresponding mutant type NEAT1 or 3'UTR of ZEB2 lacking the binding sites of miR-506-3p was also cloned into the pmirGLO vector to form the reporter vectors, NEAT1-mut and ZEB2-3'UTR-mut, respectively. The PANC-1/GR or SW1990/GR cells were seeded into 24-well plates and co-transfected with either 50 nM miR-506-3p mimics or NC using Lipofectamine 2000, together with 100 ng of the indicated luciferase reporter vector. After 48 h, luciferase activities in the transfected cells were detected using the Dual Luciferase® Reporter Assay kit (Promega, Madison, USA), according to the manufacturer’s protocol. The firefly luciferase activity was measured and normalized based on Renilla luciferase activity.
In vivo experiments
Twelve five-week-old female BALB/c nude mice (SJA, Hunan, China) were randomly assigned to two groups (six mice per group) and subcutaneously inoculated with 1×107 PANC-1/GR cells. We allowed one week for the formation of palpable subcutaneous tumors and then intraperitoneally injected the mice with gemcitabine (50 mg/kg) weekly. Tumor sizes were recorded weekly after gemcitabine treatment using the formula: width2 × length/2. Mice were sacrificed at the last measurement, the tumors were weighed, and subsequent assays were performed. All the animal experiments were performed in accordance with the experimental animal use guidelines of the National Institutes of Health and approved by the Ethics Committee for Animal Experiments of the Second Affiliated Hospital of Nanchang University.
Transferase dUTP nick end-labeling assay
In situ, cell apoptosis was detected using a One Step Transferase dUTP nick end-labeling (TUNEL) apoptosis assay kit (Beyotime, Shanghai, China) on formalin-fixed, paraffin-embedded tissues of nude mouse xenografts, according to the manufacturer's instructions. The tissues were then photographed using an inverted fluorescence microscope (Nikon, Tokyo, Japan).
Statistical analysis
All results have been expressed as mean ± SD and analyzed using SPSS statistical software (version 24.0; Chicago, USA). Statistical analysis was performed using two-tailed Student's t-test or one-way ANOVA to identify significant differences between two or multiple groups. All assays were performed independently three times. P≤0.05 was considered statistically significant.