Transcriptome sequencing data of 181 samples were generated by R package in TCGA-PAAD dataset obtained from TCGAbiolinks (https://bioconductor.org/packages/release/data/experiment/vignettes/TCGAbiolinksGUI.data/inst/doc/vignettes.html). Differential mRNA abundance analyses were carried out using DESeq2 (http://www.r-project.org/). Genes with reads < 5 in any sample were filtered out from the final quantitative analysis. Heatmap and volcano plot were constructed by normalized gene expression via R package. The normalized gene expressions were subjected to Gene Set Variation Analysis (GSVA, a non-parametric, unsupervised method for estimating variation of gene set enrichment through the samples of an expression dataset). R package survival was used for overall survival analysis. Cox proportional hazard (PH) model was executed by the functions of survival and survminer in R package. The best-scanned cutoff points are defined as the one with the most significant (log-rank test) split. R package survival ROC was used for Receiver Operating Characteristic (ROC) curve and Area Under Curve (AUC) plotted for different durations of survival analysis.
Ethical approval was obtained from the Ethical Committee of the First Affiliated Hospital of China Medical University (a tertiary hospital and regional cancer center in Shenyang, China). Written informed consent was obtained from all patients at admission. The diagnosis of pancreatic cancer was based on the National Comprehensive Cancer Network Guidelines (NCCN 2018), and no patient received preoperative chemotherapy. Pancreatic cancer tissues and adjacent normal tissue from the same patients were obtained during pancreaticoduodenectomy at the First Affiliated Hospital of China Medical University from June 1, 2018 to May 31, 2019. Tissue samples were cryopreserved in liquid nitrogen immediately after surgical resection until further experiments.
All experiments were performed with mycoplasma-free cells. Human pancreatic adenocarcinoma cell lines PANC-1 (RRID:CVCL_0480), BxPC-3 (RRID:CVCL_0186), and SW1990 (RRID:CVCL_1723) were purchased from Shanghai Zhong Qiao Xin Zhou Biothechnology Co., Ltd (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS, Gibco), 10 mM HEPES (Gibco), 2 mM L-glutamine (Gibco), 1 mM pyruvate sodium (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco) at 37°C with 5% CO2.
Stable lncRNA-knockdown cell line construction
A specific SW1990 cell line (SW1990-LNC-KD) with stable knockdown of lncRNA AL161431.1 or control SW1990 cell line was constructed using a lnc-shRNA sequence targeting 5’- GCAGTATTCCTGCACTTCT -3’ or scramble control sequence 5’- TTCTCCGAACGTGTCACGT -3’ cloned into the LV3(H1/GFP&Puro) vector (Figure S1), respectively, and packaged with lentivirus (Shanghai GenePharma, China). Cells containing the lnc-shRNA were selected by media containing 5 μg/ml puromycin (Sigma, St. Louis, MO).
Transfection of siRNAs
Short interfering RNAs (siRNAs) and scrambled negative control for lncRNA were provided by Shanghai GenePharma (Shanghai, China). The siRNAs (sequence listed in Table S1) were transfected with X-tremeGENE siRNA transfection reagent (Roche Applied Science, Shanghai, China) according to the manufacturer’s manual.
Total mRNA extraction and qRT-PCR
Fresh cells or frozen tissues were homogenized in TRIzol reagent (Thermo Fisher Scientific, Inc.) for total mRNA extraction following the manual’s instructions. The purified mRNAs were quantified by a NanoDrop 2000 Spectrophotometers (Thermo Fisher Scientific, Inc.), reverse transcribed using an RT reagent Kit (Nachuan Bio-Tech Co., Binzhou, China) based on the manufacturer’s instructions. qRT-PCR assays were performed using SYBR Green Master Mix (Nachuan Bio-Tech Co.) in an Exicycle 96 Real-Time Quantitative Thermal Block (Bioneer) according to the manufacturer’s protocol (sequence of primers listed in Table S1). The average of triplicate qRT-PCR results of target lncRNA expression from each sample was normalized by β-actin of the same sample, and the relative expression was calculated using 2−ΔΔCt.
Cell count kit-8 assay for cell proliferation
Cell proliferation was detected using a CCK-8 assay kit (DOJINDO, Japan). Cells were seeded into 96-well plates (1 x 104 /well). At 10 am each day, 10 μl CCK-8 reagent in 100 μl medium was added to each well. The absorbance at 450 nm of each well was measured after 2 hour incubation with a microplate spectrophotometer.
Forty-eight hours after siRNA transfection, cell death/apoptosis or cell cycle were analyzed by flow cytometry (LSR, BD Biosciences) using Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Shanghai, China) or Propidium Iodide (PI; Solarbio Biotech, China), respectively, according to the manufacturer’s instructions. For cell death/apoptosis, cells were collected, washed three times with cold PBS, stained in 500 μl staining buffer (Annexin V-FITC/PI in PBS) at room temperature for 30 min in dark. For cell cycle analysis, cells were collected, washed three times with cold PBS, fixed in precooled anhydrous ethanol at 4°C for 30 min, stained with 50 ug/mL PI in 500 μl PBS. All experiments were triplicated.
Cells were grown on sterile glass slides, fixed with 4% paraformaldehyde for 30 min, blocked with 1% BSA for 30 min, incubated with primary antibodies against CDH1 (E-cadherin, AF0131, 1:500; Affinity Biosciences LTD.), CDH2 (N-cadherin AF4039, 1:200; Affinity Biosciences LTD.), and VIM (Vimentin AF7013, 1:250; Affinity Biosciences LTD.) at 4°C overnight, followed by incubation with Alexa Fluor® 594 conjugated Affinipure Goat Anti-Rabbit IgG (H + L) secondary antibodies (Jackson ImmunoResearch Laboratories, PA) for 1 hour. Nuclei were counterstained with DAPI. Images were captured with Olympus IX81 inverted fluorescence microscope (Olympus, Beijing, China).
Anti-beta actin (AF7018, 1:3000), anti-E-cadherin (AF0131, 1:10000), anti-Vimentin (AF7013, 1:1000), anti-N-cadherin (AF4039, 1:1000) were obtained from Affinity Biosciences LTD., and the secondary antibody concentration was 1:5000 (S0001, Affinity Biosciences LTD.). Forty-eight hours after siRNA transfection, cells were lysed in RIPA lysis buffer (Merck Group, Germany) for 30 min on ice. Sample protein concentrations were quantified using a BCA assay kit (Solarbio, China). Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with the antibodies of interest. The intensity of bands was quantified using ImageJ, using β-actin as internal loading control.
Xenograft tumor growth in nude mice
Animals were properly treated in accordance with the institutional ethical requirements of experimental animals. Male nude mice were kept in a temperature-controlled specific-pathogen-free animal laboratory, with a 12h light/12h dark cycle. All animals had free access to food and water. SW1990-LNC-KD cells (1.5 × 106 cells in 0.1 ml sterile PBS, with stable knockdown of lncRNA AL161431.1), or SW1990-LNC-NC cells (with scrambled shRNA) were subcutaneously injected into the left flank of mice at 8 weeks of age. The volume of tumor was measured every morning by length x width x depth in mm. The mice were euthanized 2 weeks after injection, and the growth of subcutaneous tumors were compared (n = 7 in each group).
PCNA immunohistochemistry staining were performed on 4 mm sections of paraffin-embedded tissue samples. In brief, the slides were incubated in PCNA antibody (AF0239, Affinity Biosciences LTD.; at 1:100 dilution) at 4°C overnight followed by the secondary antibody (S0001, Affinity Biosciences LTD.; at 1:200 dilution) at 37°C for 1 hours. Nuclei were counterstained with hematoxylin. PCNA positive cells were quantified using IHC Profiler .
Twenty-four hours after transfection, SW1990 (1x105) or BxPC-3 (1x105) cells were resuspended in 100 µl serum-free medium and seeded into the upper chamber with 12 µm pore polycarbonate membranes pre-coated with Matrigel Basement Membrane Matrix (BD Biosciences, Bedford, MA). The lower chamber was filled with 600 μl of 1640 medium supplemented with 20% FBS. After 24 hours of incubation at 37°C with 5%CO2, cells remaining in the upper membrane surface were removed with a cotton swab, whereas invaded cells were fixed and stained with 0.5% crystal violet .
Twenty-four hours after transfection, cells were seeded in 24-well plates at 1x105 cells/well. Cells were cultured in medium containing 5% FBS with 5% CO2 for 24 h. A 1-mm wide scratch was made in the confluent cultures with a pipette tip, followed by wash twice with PBS to remove debris. The area of the scratch was measured using images taken by a phase-contrast microscope .
Statistical analysis was performed using SPSS software (version 24, Armonk, NY: IBM Corp.). Quantitative data are presented as mean ± SD. Differences in the mean of two samples were analyzed by Student’s t-test. All 2-tailed statistical tests were considered significant when p < 0.05.
Data will be available by contacting the corresponding author.