Bioinformatic analysis. Xiantao bioinformatics tool (https://www.xiantao.love/products) is an online visualized bioinformatics analysis tool that can analyze RNA expression data of tumor samples and normal samples shared by TCGA and Genotype-Tissue Expression (GTEx) projects. We first performed an unpaired analysis of PAFAH1B3 mRNA expression in 33 cancers including PAAD whereas paired analysis of PAFAH1B3 expression in PAAD. In addition, we also used the Xiantao bioinformatics tool to analyze the correlation between KLF9 and PAFAH1B3 in PAAD.
Sample collection. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. All samples were used in accordance with the statutes of the Declaration of Helsinki. Paraffin sections of pancreatic cancer tissues and their paired paracancerous pancreatic tissues from 85 patients with PDAC confirmed by pathology were obtained from our hospital. In addition, fresh cancer tissues and adjacent pancreatic tissues from 10 patients with PDAC were collected. None of the patients received any neoadjuvant chemoradiotherapy, immunotherapy or targeted therapy before surgery to exclude the impact of other treatments on the specimen study.
Immunohistochemistry (IHC)
The ethics and informed consent involved in the human tissue experiments are detailed in the “Ethical approval” section. IHC was used to detect PAFAH1B3 and KLF9 expression in PDAC tumour tissues. The specific experimental procedures are described in[25]. The primary antibodies used in the experiment were rabbit anti-PAFAH1B3 (1:100, Abcam, Cambridge, MA, USA) and rabbit anti-KLF9(1:100, Abcam, Cambridge, MA, USA). The secondary antibody was goat anti-rabbit IgG (1:500, Abcam, Cambridge, MA, USA). The staining intensity and percentage of PAFAH1B3-positive tumour cells were assessed. The intensity of the staining was evaluated by using the following criteria: 0, negative; 1, low; 2, medium; and 3, high. The extent of staining was scored as 0, 0% stained; 1, 1-25% stained;2, 26-50% stained; and 3, 51-100% stained. The final scores were calculated by multiplying the scores of the intensity with those of the extent and dividing the samples into four grades: 0, absent (-);1-2, weak (+); 3-5, moderate (++); and 6-9, strong (+++). In this study, (-~+) was defined as low PAFAH1B3 expression and (++~++) was defined as high PAFAH1B3 expression based on IHC scores.
Haematoxylin and eosin (H-E). After the slices were deparaffinized and rehydrated, haematoxylin was added to stain the nucleus for 5 minutes, and then the sections were rinsed with tap water. Eosin staining was added for 1 minute, and then the sections were rinsed with tap water. Then, the samples were dehydrated, cleared and sealed. The cells were observed and photographed under an inverted microscope.
Western blotting. Tissues and cells were lysed using RIPA lysis buffer (Solarbio, Beijing, CHN) containing protease inhibitors to extract proteins. Western blotting was performed as we reported previously[26]. The results were analysed using ImageJ. The following antibodies were used: rabbit PAFAH1B3 polyclonal antibody (1:1000, Abcam, Cambridge, MA, USA), mouse β-actin polyclonal antibody (1:1000, Boster Biological Technology, Ltd), rabbit PCNA polyclonal antibody (1:1000, Abcam, Cambridge, MA, USA), rabbit E-cadherin polyclonal antibody (1:1000, Boster Biological Technology, Ltd), rabbit N-cadherin polyclonal antibody (1:1000, Boster Biological Technology, Ltd), rabbit snail1 polyclonal antibody (1:1000, Abcam, Cambridge, MA, USA), rabbit MMP2 polyclonal antibody (1:1000, Abcam, Cambridge, MA, USA), rabbit KLF9 polyclonal antibody (1:1000, Abcam, Cambridge, MA, USA),and mouse vimentin polyclonal antibody (1:1000, Boster Biological Technology, Ltd.). Rabbit (1:10,000, Beyotime, Shanghai, China) and goat IgG (1:10,000, Beyotime, Shanghai, China) secondary antibodies were used.
qRT-PCR.Total RNA was extracted from cells using TRIzol (Solarbio, Beijing, CHN). qRT‒PCR was performed as we reported previously[26]. The relative mRNA expression was calculated with the 2−∆∆Ct method. GAPDH was used as the internal control. The primers used were as follows:
PAFAH1B3 forward primer 5′-CTGGGCTACACACCTGTTTGC-3′
PAFAH1B3 reverse primer 5′-GGAGAGTTTAATGTTGTGGGAAGG-3′
GAPDH forward primer 5′-GAACGGGAAGCTCACTGG -3′
GAPDH reverse primer 5′-GCCTGCTTCACCACCTTCT-3′
Cell culture. The human normal pancreatic ductal epithelial cell line HPDE6-C7 and human pancreatic cancer cell lines (SW1990, MIA Paca-2, BxPC-3, PANC-1, CFPAC-1, and AsPC-1) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured at 37°C and 5% CO2 in a humidified atmosphere incubator. The cells were cultured in DMEM (Doctor DE Biological, Wuhan, China) supplemented with 10% fetal bovine serum (FBS; Gibco) and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). The medium was changed every 2 days, and the growth of the cells was observed under an inverted microscope.
Plasmid construction and lentiviral transfection. Plasmids containing short hairpin RNA (shRNA) targeting PAFAH1B3 and the negative control (shNC) were constructed by Hanbio Tech (Shanghai, China). The sequence of shPAFAH1B3 was 5′-CACCATCAGCCATCATGACATGTAT-3′. The sequence of the shRNA negative control (shNC) sequence was 5′-TTCTCCGAACGTGTCACGTAA-3′. The overexpressed PAFAH1B3 plasmid was synthesized according to transcript NM_002573.4 by Hanbio Tech (Shanghai, China). Finally, these plasmids were packaged into lentiviral particles and transferred into SW1990 and MIA Paca-2 cell lines. The experimental procedures were performed as previously described[27].
siRNA and overexpression plasmid transfection siKLF9 and siNC were constructed by Hanbio Tech (Shanghai, China). The sequence of siKLF9 was 5′-GCUUGUUGGACCUGAACAAGUTT-3′, and the sequence of siNC was 5′-UUCUCCGAACGUGUCACGUTT-3′. Following the manufacturer's instructions, siKLF9 or siNC was transfected into pancreatic cancer SW1990 and MIA Paca-2 cells using EL transfection reagents. The KLF9 overexpression plasmid pcDNA3.1-KLF9 (transcript number: NM_001206.2) and the negative control pcDNA3.1 were constructed by Hanbio Tech (Shanghai, China). pcDNA3.1-KLF9 or pcDNA3.1 was transferred into pancreatic cancer SW1990 and MIA Paca-2 cells using PEI 40K transfection reagents.
CCK-8 cell proliferation assay. Cell viability was determined with a CCK-8 assay. PDAC cells were seeded into 96-well plates at 4×103 cells per well and incubated for 0 hour, 24 hours, 48 hours, and 72 hours. Then, 10 µl of CCK-8 reagent (Doctor DE Biological, Wuhan, China) was added to each well. After 1 hour of incubation at 37 °C in the dark, the absorbance was measured at 450 nm using an iMark microplate reader (Bio-Rad). Five duplicate wells were measured for the experimental groups and control groups, and the measurements were averaged.
Transwell Assay. The invasion ability of GC cells was analysed with a 24-well Transwell chamber (Biyuntian Biological Technology Co., Ltd. Shanghai, China). Transwell chambers precoated with Matrigel (YB356234, BD Biosciences, United States) were used for the invasion assay, and chambers without Matrigel were used for the migration assay. Prior to cell collection, the Matrigel gel was diluted with a serum-free culture medium at a ratio of 1:10. Next, 60 μl of diluted Matrigel was added to the upper chamber, which was placed in a 24-well plate at 37 °C for 2–3 h until curding. Next, the transfected cells (2×104 cells) in 200 μl serum-free DMEM were added to the upper chamber, and 600 μl DMEM containing 10% FBS was added to the lower chamber. After being cultured for 48 h at 37°C, the PDAC cells in the chamber were fixed with 4% formaldehyde for 15 minutes at room temperature and stained with 0.1% crystal violet at room temperature for 15 minutes.The cells on the upper surface of the chamber were gently wiped off with a thin cotton swab. Cell migration or invasion was measured and photographed under an inverted microscope.
Scratch Assay. First, the pancreatic cancer SW1990 and MIA Paca-2 cells were plated into 6-well plates and cultured until they reached 80–90% confluency. Then, the tip of a 100 µl pipette was used to scratch the monolayer. The floating cells were washed with PBS for removal. Then, fresh medium with no FBS was added, and culturing was continued for 24 hours. At 0 and 24 hours after cell scratching, cell migration was observed at the same site under a microscope, and the scratch area was measured using ImageJ.
Colony formation assay Cells were plated in 6-well plates at a density of 500 per well, and DMEM medium containing 10% FBS was added and cultured for approximately 10-14 days. Then, thee cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Subsequently, the colonies were dyed with crystal violet for 10 min and then rinsed with water to count the number of colonies formed.
EdU assay. The EdU Cell Proliferation Kit with Alexa Fluor 647 (Beyotime, Shanghai, CHN) was used to evaluate cell proliferation. Pancreatic cancer cells were incubated with 5-ethynyl-20-deoxyuridine (EdU) for 4 hours and subsequently processed according to the manufacturer’s instructions. The experimental procedures were performed as previously described[28].
In vivo experiment. The ethics involved in animal experiments are detailed in the “Ethical approval” section. BALB/C nude mice aged 5–6 weeks were purchased and housed in a sterile environment with controlled temperature and light. Tumorigenicity experiment. Nude mice were randomly divided into 4 groups with 6 mice in each group. Specific groups are as follows: SW1990 cells stably transfected with NC or PAFAH1B3-Flag; MIA Paca-2 cells stably transfected with shNC or shPAFAH1B3. Each group of cells was collected and resuspended in 100 µl DMEM, with approximately 5x106 cells. The volume and weight of the tumours were determined every 3 days. Four weeks after inoculation all nude mice were sacrificed by CO2 inhalation, and the subcutaneous tumours were collected and measured. The expression of Ki-67 in tumours of each group was detected by IHC. Metastasis experiment: Nude mice were randomly divided into 2 groups with 6 mice in each group. MIA Paca-2 cells transfected with PAFAH1B3-Flag or NC were resuspended in 100 µl DMEM, with approximately 2x106 cells in each group. The liver metastasis model of pancreatic cancer was established by spleen inoculation in nude mice[29]. Then, MIA Paca-2 cell suspensions were injected into the spleens of nude mice to establish a liver metastasis model. All nude mice were sacrificed by CO2 inhalation 4 weeks after inoculation to observe liver metastasis, and the differences between the two groups were compared.
Chromatin immunoprecipitation (ChIP) assay. ChIP assays were performed with an EZ-ChIP Kit (Millipore) according to the manufacturer’s protocol. Briefly, the pancreatic cancer lines SW1990 and MIA Paca-2 were cross-linked with 1% formaldehyde and terminated after 5 minutes by the addition of glycine. Cells were harvested with SDS lysis buffer and were sheared by sonication cycles to generate DNA fragments with an average size of 200–1000 bp for qChIP. Preclearing and incubation with KLF9 or IgG control overnight was performed, followed by incubation with protein-A/G beads for 2 hours at 4°C. Washing and reversal of crosslinking were performed. The immunoprecipitated DNA was extracted with phenol-chloroform followed by ethanol precipitation. Purified DNA was analysed by quantitative real-time PCR (qPCR), and the enrichment was expressed as fold enrichment compared with IgG.
Dual-luciferase reporter assay. ThePAFAH1B3 promoter sequence h-PAFAH1B3-pro-WT and mutant sequence h-PAFAH1B3-pro-MUT were constructed, and the sequences were constructed on the pGL3-basic vector containing firefly fluorase. The KLF9 overexpression plasmid pcDNA3.1-KLF9 and the overexpression control plasmid pcDNA3.1-NC were constructed. Reninase was inserted into the PRL vector.The details are grouped as follows:
Group 1, h-PAFAH1B3-pro-WT+ pcDNA3.1-NC+PRL;
Group 2, h-PAFAH1B3-pro-MUT+ pcDNA3.1-KLF9+PRL;
Group3,h-PAFAH1B3-pro-WT+pcDNA3.1-NC+PRL; Group4,h-PAFAH1B3-pro-MUT+ pcDNA3.1-KLF9+PRL.
After each sequence was transferred into 293T cells for 48 h, the cells of each treatment group were analysed by double luciferase. Relative fluorescence ratio =Firefly luciferase/Renilla luciferase
Statistical analysis. SPSS24.0 (IBM SPSS Statistics, Armonk, NY) statistical software and GraphPad Prism 8.0 (Inc., San Diego, CA, USA) were used for statistical analysis and mapping of the data. Measurement data are represented by the mean ± standard deviation, and differences between groups were compared by T test or nonparametric test. When more than two groups were compared, one-way analysis of variance (ANOVA) was used to analyse the differences. All experiments were performed in triplicate. The chi-square test was used to detect the relationship between PAFAH1B3 expression and clinicopathological parameters. The Kaplan‒Meier test was used for survival analysis. The Spearman method was used for correlation analysis. In all analyses, *, ** and *** indicate P<0.05, P<0.01 and P<0.001, respectively.
Ethical approval. For human tissues: All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University, China. All of the human tissues used in the present study were obtained with written informed consent from all subjects and their legal guardians. For animals: All experimental protocols were approved by the Animal Ethics Committee of the Second Affiliated Hospital of Nanchang University, China. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).