Human specimens
Eighty-four pairs of cancerous and adjacent normal tissues were collected from patients diagnosed with pancreatic cancer from 2015 to 2017 at the First People’s Hospital of Changzhou. All the specimens were frozen in liquid nitrogen and stored at − 80 °C for further use. The study was approved by the Ethics Committee of the First People’s Hospital of Changzhou and written informed consent was signed and returned by each patient before using the tissues and clinical data.
Cell culture
Human pancreatic duct epithelial cell Line (HPDE6-C7), and human pancreatic cancer cell lines CAPAN-1, BxPC-3, SW 1990 and PANC-1were purchased from ATCC (Manassas, USA). CAPAN-1 and BxPC-3 cells were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific). PANC-1cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (Gibco Thermo Fisher Scientific). SW 1990 cells were cultured in Leibovitz’s L-15 medium and HPDE6-C7 was maintained in Keratinocyte Serum Free Medium supplemented with 25 mg/500 ml Bovine Pituitary Extract and 2.5 µg/500 ml Epidermal Growth Factor (Gibco, Thermo Fisher Scientific). They were all kept at 37 °C in a humidified atmosphere containing 5% CO2.
Oligonucleotides and transfection
The small interfering RNAs (siRNAs) for DLX6-AS1 [siDLX6-AS1 and siDLX6-AS1(a)] and the respective negative control siRNA, miR-181b mimics (5′-AACAUUCAUUGCUGUCGGUGGGU-3′), miR-181b inhibitors (5′-ACCCACCGACAGCAAUGAAUGUU-3′) and the respective negative control miRNAs were purchased from RiboBio (Guangzhou, China). Cell transfections (final concentration for siRNA transfection: 100 nM, final concentration for miRNAs transfection: 50 nM) were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol and the transfection efficiency was confirmed using quantitative real-time PCR (qRT-PCR). All the experiments were performed in triplicate.
RNA isolation and qRT-PCR
Total RNA was extracted from tissue or cells through TRIzol reagent (Invitrogen, Carlsbad, USA). The extracted RNA was then reversely transcribed into cDNA using PrimeScript™ 1st strand cDNA Synthesis Kit (Takara, Dalian, China). Real-time PCR was performed with SYBR Premix Ex Taq (Takara, Dalian, China) in the ABI7500 system. GAPDH or U6 was used as internal control for data analysis. All the experiments were performed in triplicate.
CCK-8 assay
For proliferation assay, the transfected cells were seeded in 96-well plates at the density of 5000 cells/well over night. At 0, 24, 48 and 72 h after transfection, the cells were incubated with 10 µl CCK-8 reagent for 2 h at 37 °C in the dark, and the OD value at 450 nm was read using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) to determine the viability. All the experiments were performed in triplicate.
Colony formation assay
The transfected cells were seeded in a 6-well plate at the density of 1000 cells/well and incubated in full culture medium at 37 °C. At 14 day, the cells were washed with PBS, fixed with methanol and stained with 1% crystal violet. The number of colonies was counted under a microscope. All the experiments were performed in triplicate.
Transwell migration and invasion assays
Transwell migration and invasion assays were measured by Transwell chamber (8 µm pore size, Corning) without Matrigel for migration assay and with Matrigel for invasion assay. At 48 h after transfection, 1 × 105 cells were cultured in the upper chamber with serum-free median. The lower chamber was filled with median containing 10% FBS as the chemoattractant. After incubation for 48 h, cells in the upper membrane were removed with cotton swab while those that migrated or invaded were fixed in methanol, stained with 0.1% crystal violet and quantified under a microscope. All the experiments were performed in triplicate.
Luciferase reporter assays
DLX6-AS1 fragments with the wild type (WT) or mutant (MUT) miR-181b binding sites were cloned to generate the plasmids pmirGLO-DLX6-AS1-WT or pmirGLO- DLX6-AS1-MUT (Promega, Madison, USA). The 3′UTR of ZEB2 containing the wild type or mutant miR-181b binding sites were used to generate the plasmids pmirGLO-ZEB2-WT and pmirGLO-ZEB2-MUT (Promega). HEK293T cells were co-transfected with luciferase plasmids and miR-181b mimics, or mimics NC by using Lipofectamine 2000 reagent according to the manufacture’s instruction (Invitrogen). At 48 h after transfection, firefly and Renilla luciferase activities were measured by a Dual-Luciferase Reporter Assay System (Promega) and the experiments were performed in triplicate.
Western blot
The tissue or cells were collected and total proteins were extracted and quantified. Equal amount of proteins was subjected to 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore, Bedford, USA). After blocking with 5% fat-free milk, the membranes were probed with primary anti-ZEB2, anti-E-cadherin, anti-vimentin or anti-N-cadherin antibodies (Abcam, Cambridge, USA) at 4 °C overnight. Then membranes were incubated with horseradish peroxidase-conjugated IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA) at 37 °C for 2 h. Enhanced chemiluminescence kit (Pierce, Waltham, China) with imaging system (Bio-Rad, Hercules, USA) were used to analyze the protein signals and β-actin was used as the internal control.
Tumor growth and tumor metastasis assay in nude mice
Lentivirus expressing shDLX6-AS1 or shRNA control were designed and packaged by Genechem (Shanghai, China), and stable cell lines were established by infecting lentivirus into SW1990 cells and selected by puromycin (Sigma, St. Louis, USA). All the animal experiments were approved by the Animal Experimentation Ethics Committee of the First People’s Hospital of Changzhou Hospital. For the tumor growth assay, twelve female BABL/c athymic nude mice (4–5 weeks old) were subjected to flank subcutaneous injection of SW1990 cells stably expressing shDLX6-AS1 or shNC. Tumor volume was measured every 5 day from the 10 day to the 35 day after inoculation. The volume was calculated as: volume = (length × width2)/2. At 35 day, the mice were sacrificed and the tumors were removed, weighed and snap-frozen for RNA extraction. For the tumor metastasis assay, twelve female BABL/c athymic nude mice (4–5 weeks old) were subjected to tail vein injection of SW1990 cells stably expressing shDLX6-AS1 or shNC. At 35 day, all the mice were sacrificed and the lung were excised, and the number of metastatic nodules in the lung was counted.
Statistical analysis
Data were presented as mean ± standard deviation. The data analysis was performed by using GraphPad Prism software (Version 5.0, GraphPad, San Diego, USA). The significant differences in the clinical parameters between low expression and high expression of DLX6-AS1 groups were analyzed by Chi square test. Significant differences for the mean values between groups were determined by the Student’s t test or one-way ANOVA, as appropriate. P value < 0.05 was considered statistically significant.
