We downloaded Gene expression profile GSE67549 and GSE9350 from Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/gds). GSE67549 contained 9 normoxic PC cells samples and 9 hypoxic PC cells samples, while GSE9350 contained 2 normoxic PC cells samples and 2 hypoxic PC cells samples. Different expression genes were identified using the cut-off as Log2 fold change (FC) >1 and P value <0.05. Common differently expression genes were analyzed using intersection analysis. The expression of these genes in the PC samples of The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) database was determined using GEPIA (http://gepia.cancer-pku.cn/), P<0.05 was a threshold to consider significant.
Total 90 paired PC tissues and adjacent pancreatic tissues were provided by the PC patients which obtained surgery in the Affiliated Hospital of Guizhou Medical University. None of them obtained radiotherapy or chemotherapy prior to the surgery. The current study was approved by the Ethics Committee of Guizhou Medical University in accordance with Declaration of Helsinki, and patients who participated in the current study all obtained and signed the received informed consent.
Cell culture and transfection
Two human PC cell lines (PANC-1 and AsPC-1) used in the current study were obtained from ATCC. PANC-1 and AsPC-1 cells were incubated in DMEM with 10% FBS at the 37˚C environment with 5% CO2. The condition of normoxia was set to 21% O2 and 5% CO2, while the condition of hypoxia was set to 1%O2, 94%N2 and 5%CO2. The FUT11 interference lentivirus and blank lentivirus plasmids were obtained from Sangon Biotech (Shanghai, China). PDX1 overexpression lentivirus was purchased from GeneChem (Shanghai, China). The small interfering RNAs (siRNAs) targeting HIF1α were obtained from JIMA (Shanghai, China). To construct the stable cell lines with target gene overexpression or knockdown, 1 μg/mL puromycin (Sigma, USA) was added to continuously screen after transfection with lentivirus at 48h for 10 days.
Quantitative real-time fluorescent PCR
Total RNA in PC tissues and cells was separated using TRIZOL (Beyotime Biotechnology, Hangzhou, China) and diluted into DNase/RNase-free water. After quantify, total RNA (2µg per sample) was reversely transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA). Finally, qPCR was conducted to determine the expressed level of target genes using SYBR™ Green PCR Master Mix (Solarbio, Wuhan, China). β-actin was set to the control. The primer sequences in our study were purchased from Tianyi Huiyuan (Wuhan, China) and shown as Table 1.
Cell counting kit-8 (CCK-8) assay
PANC-1 and AsPC-1 cells were plated in a 96-well plate in sextuplicate with 3×103 cells/well. Brief, 100μl DMEM medium contain 10μl CCK-8 regent (Boster, Wuhan, China) was added to each well in 24h, 48h, 72h and 96h respectively. The absorbance of each well was detected at 450nm.
5-ethynyl-2’-deoxyuridine (EDU) assay
The EDU assay was carried out using a BeyoClick™ EdU-488 Proliferation Detection Kit (Beyotime, Suzhou, China). In brief, while PC cells adherence in 6-well plate, primary medium was removed and fresh medium were added. Then, total 10μM EDU was injected into each well and cells were cultured in 37°C for 2.5h. Following by fixation using 4% paraformaldehyde (Beyotime, Suzhou, China) for 15 min and permeabilization using 0.3% Triton X-100 (Boster, Wuhan, China) for 8 min, total 500μl Apollo dyeing reaction buffer was then added for 40 min in dark environment. After staining the nuclear using DAPI for 10 min, the proportion of EDU was detected using fluorescence microscopy.
Colony formation assay
Cell with a density of 2000 cells/well were seeded into 6-well plates and cultured in DMEM media containing 10% FBS. After 24h, intervention factor was added and cultured for 2 weeks. After fixation in 4% paraformaldehyde for 15 min, 1% crystal violet was used to stain cell colonies. Cell colonies was counted and photographed.
The proteins in PC cells and tissues were extracted using RIPA reagent contain 5% PMSF protease inhibitor. The BCA method was performed to detect the protein concentration of each sample. Proteins (30μg/ per line) were added and separated by 10% SDS-PAGE for 120 min. Then, the proteins were transferred into the PVDF membranes (Millipore, USA) with 0.45μm bore diameter, which was then seted in the environment contained 5% BSA for 30min and incubated with primary antibody including FUT11, N-cadherin, E-cadherin, PDK1, HIF1α and β-actin (all purchased from CST company) for 12h in 4˚C. High sensitivity ECL reagent was used to visualize the blots in MultiImager and the relative expression of protein was calculated using Image J. β-actin was set as reference for FUT11, N-cadherin, E-cadherin, PDK1 and HIF1α.
For transwell migration assay, total 4´105 cells were suspended using 200μl DMEM medium without FBS and seeded into the upper transwell chambers (Becton, Dickinson and Company, USA). 600μl DMEM medium contained 10% FBS was placed in the lower transwell chambers. After 28h, migratory cells were fixed with paraformaldehyde and stained using 0.5% crystal violet. The transwell invasion experiment required matrigel(Becton, Dickinson and Company, USA) to be coated in the upper chamber before cell seeding. After fixation using 4% paraformaldehyde, staining with 1% crystal violet (Boster, Wuhan, China) and removing non-invasion cells using cotton swab. Finally, chambers were was photoed and counted.
In vivo assay
For subcutaneously injected model, total 10 female BALB/c nude mice were obtained from the animal central of Guizhou Medical University (Guizhou, China). After adaptive feeding, total 1×107 PANC-1 cells with FUT11 knockdown and negative control cells were subcutaneously injected into upper-right flank of BALB/c mice(n = 5 in each group). The health status of mice was monitored per day, while the tumor volume was detected per week. The tumor volume was monitored once a week and determined as followed: (mm3) = (Long×Width2 )/2. After 5 weeks. The protein level of KI67 and PCNA in tumor tissues was determined using immunohistochemical staining. For the metastasis assays, the metastasis ability of FUT11 knockdown and negative control group PANC-1 cell using spleen capsule injected liver metastasis model. FUT11 knockdown and negative control group PANC-1 cells (1´ 107 cells ) were injected into the spleen of BALB/c mice (n=5 in each group). Mice were sacrificed 12 weeks after inoculation, the liver tissues were harvested and photoed. HE stain was used to detected the metastasis loci in the liver. All procedures of animal studies in the current study were approved by Ethics Committee of Guizhou Medical University and conformed to legal mandates and national guidelines for the care and maintenance of laboratory animals.
Cells were fixed with 4% paraformaldehyde (Solarbio, Wuhan, China) for 15min. Then, the samples were cultured with anti-FUT11 (mouse source) , anti-N-cadherin(rabbit source), anti-E-cadherin(rabbit source), anti-HIF1α(rabbit source) and anti-PDK1 (rabbit source) primary antibody. After removing dissociative primary antibody by PBS, the samples were incubated with secondary antibodies anti-mouse conjugated FITC (Proteintech, Wuhan, China) and secondary antibodies anti-rabbit conjugated Cy3 (Proteintech, Wuhan, China). Nuclear were stained with DAPI (Boster, Wuhan, China). Finally, confocal microscopy or fluorescent microscope was used to collect the photo.
Cells were lysed in weak RIPA buffer (Proteintech, Wuhan, China) that included a 1% PMSF (Boster, Wuhan, China). The liquid was centrifuged and the protein was collected. Then, the anti-FUT11 (1:50) antibody and IgG (1:50; Beyotime Biotechnology; Hangzhou, China) was added for 6h, respectively. The, the A/G agarose beads (Boster, Wuhan, China) was added for 3h. After washing by PBS three times, isolated immunoprecipitates in beads were collected and analyzed using western blot.
After predicting binding site of HIF1α in the promoter of FUT11 using online database JASPAR(http://jaspar.binf.ku.dk/), dual luciferase reporter assay was performed to verify the bind. FUT11 promoter sequence with full‐length and truncated were carried into the psi‐basic luciferase reporter vector (Promega, USA). Finally, a total of 1×104 PANC-1 and AsPC-1 cells were plated into 24-well plate and cultured for overnight at 37 °C. Then, both of full‐length and truncated FUT11 luciferase reporter vector and the si‐HIF1α/si‐NC were co-transfected into PC cells using lipidosome 2000 (Solarbio, Wuhan, China). After PC cells were cultured to normoxia or hypoxia, Finally, the luciferase activity of cells was determined after transfection at 24h.
SPSS software (version 21.0) was employed to perform statistical analysis. The difference between two groups was performed using paired t-test, while the difference among multiple groups were determined based on one-way analysis of variance. P*<0.05 was used as cut-off to consider significant.