Patients and tumor specimens
PC and corresponding peri-tumor tissues of 60 surgical resections of patients without preoperative treatment were provided by the Institute of Pancreatic Diseases of Union Hospital (Wuhan, China) from 2013 to 2017. The Ethics Committee of the Academic Medical Center of Huazhong University of Science and Technology approved the procedure of human specimen collection (Permission No: 2013, S199), and all patients signed informed consent. The tissues were delivered to the laboratory within 20 min, and then either formalin fixed and paraffin-embedded or snap frozen as described previously .
PANC-1 and BxPC-3 cell lines were originally obtained from the ATCC (American Type Culture Collection). All cell lines were subjected to authentication using short tandem repeat multi-amplification and tested negative for mycoplasma contamination. PANC-1 cells were grown in DMEM (Gibco, USA) medium. BxPC-3 cells were grown in RPMI-1640 (Gibco, USA). All the medium contained 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin. For glucose or glutamine deprivation, cells were plated overnight in complete DMEM, briefly washed with phosphate-buffered saline (PBS) and then transferred into glucose-free DMEM (Thermo Fisher, catalogue #11966025) or glutamine-free RPMI 1640 (Thermo Fisher, catalogue #21870076) supplemented with 10% dialyzed FBS (Gibco, catalogue #26400), respectively.
Firstly, the cultured cells were digested with 0.4% trypsin and counted. Plant the cells in 96-well plate with 2000 cells per well, and set a blank control well for a total of 5 plates. From the next day, take out a 96-well plate at the same time every day. Add 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, 5 mg/ml) to the well. Place the plate in an incubator at 37°C in the dark for 4 h. Then replace the medium with 150 µl DMSO (Sigma). After the cells were completely lysed, the viability of cells was measured by measuring the absorbance at 570 nm using an ELISA reader (Thermo Fisher Scientific). The experiment above was repeated 3 times independently.
Transwell and wound healing assay
Cell invasion and migration were evaluated using Transwell inserts (Corning-Costar; pore size 8 µm) with or without Matrigel (Sigma) coating. Cells were resuspended in FBS-free media prior to addition to the upper chamber of wells containing this insert, with media containing 30% FBS added to the lower portion. Cells were then incubated for 24 h, after which those not invading were removed, and the remaining cells under the membrane were fixed and stained. The number of invasive and migrated cells in 5 random fields was then determined via light microscopy. Additionally, the wound healing experiment was utilized to measure the migration ability of cells. The cells were plated until they were confluent in the wells of the 6-well plate, and then a sterile pipette tip was employed to scrape the monolayer of cells. Cell images at 0 h and 24 h post-wounding were used to gauge migration rates.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
TRIzol® Reagent (Invitrogen) was purposed to extract total RNA from PC cells and tissues. PrimeScript RT Master Mix (RR036A, Takara) was utilized for the RNA reverse-transcribed into cDNA according to provided directions. Real-time quantitative PCR was performed using TB Green® Premix Ex Taq™ (RR420A, Takara) based on the manufacturer’s recommendations. β-actin acted as a control for cellular RNA. The primers sequences used in this research were placed in Table S1.
Ice-cold PBS was employed to wash the cells 2–3 times. After adding RIPA lysis buffer supplemented with protease inhibitor Cocktail (1:50) and phenylmethyl sulfonyl fluoride (PMSF, 1:100), protein was extracted immediately. An appropriate amount of 5×sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein loading buffer was added into the protein sample in a 4:1 ratio. Boil water for 10 min to denaturant and store at -2°C. Protein was separated using SDS-PAGE gel, subsequently transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, USA). Membranes were blocked in 5% non-fat milk for 1 h at room temperature. Then incubated with specific primary antibodies overnight at 4°C followed by the incubation of anti-mouse or rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (#7076S/7074S, CST, dilution 1:3000). Afterwards, the enhanced chemiluminescence assay (ECL, 34095, Pierce) was implemented to visualize the band signals, collected by the ChemiDoc XRS molecular imager system (Bio-Rad, USA). Primary antibodies against ZEB1 (21544-1-AP, Proteintech, dilution 1:1000), AMPK (#2757, CST, dilution 1:1000), Phospho-AMPKα (Thr172) (#50081, CST, dilution 1:1000), Phospho-AMPK Substrate Motif (#5759, CST, dilution 1:1000), Ubiquitin (10201-2-AP, Proteintech, dilution 1:1000), PAN-AC (66289-1-lg, Proteintech, dilution 1:1000), Phospho-Ser/Thr (ab117253, Abcam, dilution 1:1000), E-cad (#3195, CST, dilution 1:1000), VIM (#5741, CST, dilution 1:1000) and β-actin (20536-1-AP, Proteintech, dilution 1:1000) were used.
The Co-IP assay for AMPK and ZEB1 was conducted via lysing PANC-1 or BxPC-3 cells with Pierce IP lysis buffer (#87787, Thermo) containing protease inhibitor cocktails and PMSF on ice for 30 min followed by sonication (power 10 for three cycles, 10 s on and 50 s for rest) to release nuclear proteins completely. One tenth of the lysate was extracted as input. The remaining lysate was then incubated with IgG, ZEB1 or AMPK antibody overnight at 4°C followed by the incubation of Protein A/G Magnetic Beads (Bimake, Houston, TX, USA) at 4°C for 2 h. Then the protein/agarose complex was isolated and resuspended in loading buffer, degenerated and further analyzed by Western Blot.
The method is similar to the previous article . KangChen Bio-tech (Shanghai, China) provided the Arraystar human LncRNA microarrays V3.0. Total RNA was collected from PANC-1 cells cultured with complete medium, glucose-free medium or glutamine-free medium using TRIzol® Reagent (Invitrogen). NanoDrop ND-1000 was employed to evaluate the RNA quantity and purity. Standard denaturing agarose gel electrophoresis was conducted to assess RNA integrity. Quick Amp Labeling Kit, One-Color (Agilent), was utilized to amplify RNA and label to generate cDNA. The cDNA samples were purified and labeled with a Low Input Quick-Amp Labeling Kit (Agilent) according to the manufacturer’s instructions. After further purification, the cDNA samples were microarray hybridized at 65°C for 17 h according to the instructions of the gene expression hybridization kit (Agilent). Then a microarray scanner (Agilent) was used to scan all microarrays.
RNA immunoprecipitation (RIP)
Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was applied to perform the RIP assay according to the provided direction. Cells were lysed by the Pierce IP lysis buffer (#87787, Thermo), blended with protease inhibitor (cocktails/PMSF) and RNase inhibitors. One tenth of the lysate was extracted as input. Subsequently, Magnetic beads were pre-incubated with IgG, ZEB1 or AMPKα2 antibody separately for 30 min at room temperature. Then mixed the washed Magnetic bead antibody complex and the remaining cell lysates at 4°C overnight. The co-precipitated RNA was purified using TRIzol® Reagent (Invitrogen) according to the manufacturer’s protocol. After RT-qPCR, 2% agarose gel electrophoresis was performed to detect the precipitated RNA.
RNA pulldown assay was performed as previously described . The double-stranded DNA template was amplified by RT-qPCR. Then the ZFAS1 and antisense of ZFAS1 were transcribed using the MAXIscript® Kit (Ambion, USA) with T7 phage RNA polymerases employed to mix together with RNA polymerase, rNTPs, and transcriptional buffer for in vitro transcription with double-stranded DNA as the template. The Pierce™ RNA 3' End Desthiobiotinylation Kit (Thermo Scientific, USA) was utilized to attach biotinylated nucleotide to the 3 'terminal of ZFAS1 and antisense of ZFAS1. The Pierce™ Magnetic RNA-Protein Pulldown Kit (Thermo Scientific, USA) and MAXIscript® Kit (Ambion, USA) were used for RNA pulldown. Prepare cell lysate and extract one-tenth of it as input. The remaining cell lysates were incubated with complex of biotinylation labeled RNA and magnetic beads. After elusion, samples were analyzed using Western Blot. All the procedures were conducted as the given instructions.
Transfection of specific siRNAs for ZEB1, AMPK, ZFAS1 or negative control were purchased from GenePharma (Shanghai, China). All the overexpression plasmids of full-length ZEB1, ZFAS1 and truncations of ZFAS1 and empty vector purchased from GeneChem (Shanghai, China). For stable transfection, full-length ZEB1 and ZFAS1 siRNA were constructed into lentiviral vector, with empty vector as a negative control. Lipofectamine 2000 (Invitrogen, USA) was used for cell transfection according to the manufacturer’s protocol. The sequences of overexpression plasmids and siRNAs were placed in Table S2.
Chromatin immunoprecipitation (ChIP)
ChIP assay was performed in the directions of the ChIP assay kit (Millipore, Lot # 2983461, USA). Enough cells were first cross-linked with 1% formaldehyde for 10 min, and then the cells were lysed according to the protocol in the kit. All the procedures were conducted on ice. Ultrasonic treatment was employed to shear the DNA. ZEB1 or IgG antibodies and agarose beads were added into the lysate for immunoprecipitation reaction. The precipitated complex of protein and DNA was then eluted and purified. The isolated DNA fragments were subjected to PCR, and the PCR products were analyzed by 2% agarose gel electrophoresis.
Luciferase reporter assay
PANC-1/BxPC-3 cells were co-transfected with pRL-TK plasmid encoding Renilla luciferase and luciferase reporter plasmid encoding firefly luciferase or/and ZEB1/ZFAS1 promoter sequence (wild-type or mutant-type) for 24 h. Then the cells were cultured under glucose and glutamine depletion for 24 h. The luciferase activities were evaluated using a luciferase activity kit (Promega, USA). Renilla luciferase served as a normalizing control. The procedure was summarized as follows: the cells were digested with 0.4% trypsin and seeded into 96-well plates. On the second day, the cells were lysed using a diluted Passive Lysis Buffer. Then add the Luciferase Assay Buffer II to measure the firefly luciferase activity. Add 1XStop&Glo® Reagent to stop the reaction and measure the second fluorescence value, the Renilla luciferase. Promoter activity per well = firefly luciferase activity/Renilla luciferase activity. The experiment above was repeated 3 times independently.
The paraffin-embedded tissues were sectioned and dewaxed with xylene. Then wash with different concentrations of alcohol (100%, 95%, 80%, 70%) and distilled water respectively. Soaked in 3% hydrogen peroxide for 10 min, followed by washing with distilled water. Added citric acid buffer pH = 6 and cook in microwave for 10 min. After cooling to room temperature, wash with distilled water and PBS, add 10% goat serum drop by drop, and place it in the 37°C incubator for 30 min. After incubating with anti-ZEB1, E-Cad, VIM primary antibodies and biotin secondary antibodies, DAB solution was added on the tissues. Then hematoxylin counterstained, dehydrated, transparent, and sealed were performed. Finally, the inverted microscope was utilized to collect the image of the stained section.
BALB/c male nude mice (4 weeks old) were purchased from HFK Bio-Technology Co (Beijing, China). The mice were randomly divided into four groups (n = 6). PANC-1 cells (2×106/mice) transfected with LV-Control, LV-ZEB1 or LV-siZFAS1 were subcutaneously injected into nude mice from the tail vein. The growth of mice was observed every 1–3 days. The date of death was recorded. Visible metastases on lung were counted and displayed by hematoxylin and eosin (H&E)–stained slides. The Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) approved the study.
All experiments were conducted at least 3 times. SPSS 22.0 (IBM, USA) and GraphPad Prism 8 (GraphPad Software, USA) were utilized for all the statistical analysis. The results are presented as the mean ± S.D. analyzed by Student’s t test or one-way ANOVA. Relative gene expression was analyzed using the 2−ΔΔCt method. Survival curves were performed using the Kaplan–Meier method and assessed using the log-rank test. All the statistical tests were two-sided. The difference was considered to be significant at *, P < 0.05, **, P < 0.01, ***, P < 0.001. NS, no significant.