Analysis of pancreatic cancer gene expression data from GEO and TCGA database
The Hypoxia-related gene expression signature consisted of 200 genes and EMT-related gene expression signature consisted of 200 genes were obtained from the gene set of HALLMARK_HYPOXIA and HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION in The Molecular Signatures Database (MSigDB, https://www.gsea-msigdb.org/). Microarray gene expression data from GSE15471 and GSE16515 were used to screen for genes that were highly expressed in PC tissues and then merged with 200 hypoxia-related genes. Level3 TCGA RSEM Gene expression data and clinical information for pancreatic cancer were downloaded from UCSC (https://xena.ucsc.edu/public). We got 30 highly expressed hypoxia-related genes in pancreatic cancer tissues. Then we calculated the hypoxia score of TCGA samples using the 30 genes as previously described(17). Briefly, for each gene, samples with the top 50% of expression value were given a score of +1, and samples with the bottom 50% of expression value were given a score of –1. The hypoxia score in each sample was the sum of the scores of 30 genes (Table S1). Similarly, EMT score of TCGA samples was calculated using 200 EMT-related genes using the same method (Table S2). TCGA subtype defined by Bailey et al.(18), Collisson et al.(19), and Moffitt et al.(20) were derived from the most recent TCGA pancreatic adenocarcinoma subclassification(21).
Functional Analysis and GSEA
Spearman correlation analysis was used to find genes related to hypoxia scores (Table S2) and NOX4 expression. The R package “clusterProfiler” (22) was applied for the Gene Ontology (GO) analysis and KEGG analysis of genes positively related to hypoxia score. Hallmark gene sets were enriched using Metascape(23). Gene-set enrichment analysis (GSEA) was applied to enrich hallmark gene sets related to NOX4 in TCGA samples.
50 PC Formalin-Fixed Paraffin-Embedded (FFPE) samples were obtained from the Affiliated Drum Tower Hospital of Nanjing University Medical School (Nanjing, China) between January 2007 and August 2013. Frozen specimens of 6 PC patients and 6 benign pancreatic lesions were collected from the same hospital between 2018 and 2019. The experimental study was approved by the Affiliated Drum Tower Hospital of Nanjing University Medical School, and the informed consent forms were obtained from patients enrolled in this study.
Cell culture and treatment
Human pancreatic cancer cell lines HPAC and Panc1 were a gift from the Technical University of Munich, Germany. ALL cell lines used in this study are considered to be identical to the reference cell line in the Cell Bank STR database, as the STR profile yields a 100% match. All cell lines were cultured in DMEM medium (Wisent Inc, Montreal, Canada) with 10% FBS (Biological Industries, Beit-Haemek, Israel) under 5% CO2 at 37 ℃. HPAC cells were treated with GLX351322 (MedChemExpress, Shanghai, China) with a concentration of 0, 1, 5, 10μM for 48 hours. The cells were then used to detect CCK8. Other interventions for cells were shown in supplementary materials.
NOX4 knockdown and overexpress
NOX4 short-hairpin RNAs (shRNAs) or scrambled control shRNA were designed, synthesized and packaged into lentivirus particles (Corues Biotechnology, Nanjing, China). Cells were plated into six-well plates (3×10^5 cells per well). Before transfection, the culture medium was replaced with DMEM with 10%FBS and 1 µg/mL Polybrene (GeneChem, Shanghai, China). Infectious lentivirus particles were harvested for 72 hours after transfection.
To establish NOX4 overexpressed HPAC cells, HPAC cells were transfected with NOX4 Human Tagged ORF Clone (RC208007) and the pcmv6 empty vector (Origene, MD, USA) using lipo3000 (Thermo Fisher Scientific, MA, USA). After 72 hours, cells were treated with G418(Sigma-Aldrich Corp., MO, USA) to select stably transfected clones.
RNA extraction and RT-PCR
Total RNA was isolated from cells using RNAiso Plus Reagent (Takara, Kusatsu, Japan). RT reactions were performed using the PrimeScript™ RT Master Mix (Takara). Then quantification of mRNA expression was performed using SYBR® Advantage® qPCR Premix (Takara) in a total reaction volume of 20 µl according to the manufacturer's instructions. The reaction was performed using the LightCycler® 96 system (Roche Diagnostics, Basel, Switzerland). ACTB was used as an internal control. The primer sequences used were shown in the supplementary material.
Cell or tissue homogenate was used for immunoblot analysis. Specific information was described in supplementary materials.
Paraffin sections of tissues from 50 PC patients were used for immunohistochemical detection of NOX4 expression. The detailed protocol was presented in the supplementary material.
Cells or frozen tissue sections (15um thick) were fixed with paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 2% BSA in PBS and incubated with NOX4, CDH2, VIM, H3K4ME3 antibodies overnight at 4°C. Then the sections and cells were incubated with secondary antibody (1:500, supplementary material) for 2 hours, counterstained with DAPI (Beyotime) and visualized by a fluorescence microscope (Olympus, Tokyo, Japan).
Cell Counting Kit-8 (CCK8) assay
CCK8 assay (Dojindo, Kumamoto, Japan) was used to detect the cell viability of PC cells according to the manufacturer's instructions. HPAC cells were planted in 96-well plates (5000 cells each) and incubated with 10 μL CCK8 for 2 h at 37 °C. The absorbance was recorded at 450nm.
Migration and invasion assays
Migration and invasion assays were proceeded in transwell chamber (Corning, NY, USA) with (invasion) or without (migration) Matrigel matrix (Corning) in a 24-well plate. 5x106 cells resuspended in serum-free DMEM were added in the up chamber, and DMEM medium with 20% FBS were added in the bottom chamber. Then cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Cells were imaged and counted using a 20× microscope.
Four-week-old male BALB/c nude mice (weighing 16–18 g) were purchased from Changzhou Cavens Experimental Animal Co. Ltd (Changzhou, China). To establish the subcutaneous transplanted model, HPAC cells were injected subcutaneously to the flank region of nude mice of 6 weeks old. Tumors were measured using vernier callipers twice a week, and the volume of tumors was calculated using the formula (length x width2/2). After one month, all mice were sacrificed, and tumors were collected. For lung-metastasis xenografts, 1×106 HPAC cells suspended in 100 µL cold PBS were injected into the lateral tail vein (4 for each group). After one month, all mice were sacrificed, and lung tissues were fixed with 4% paraformaldehyde. H&E staining was used to evaluate the proportion of metastatic lesions. All animals were approved by the Ethics Committee of Nanjing Drum Tower Hospital. All animals used in this study were treated humanely and followed guidelines set by the Animal Care Committee. The study was approved by the Ethics Review Committee for Animal Experimentation at Nanjing Drum Tower Hospital (Nanjing, China).
Chromatin Immunoprecipitation(CHIP)-PCR was performed to analyze the effect of NOX4 on the binding of H3K4ME3 to the SNAIL1 promoter sequence. Detailed information was presented in the supplementary material.
All bioinformatics analyses were performed using R (https://www.r-project.org/) and Rstudio software (B Corps™, DE, USA). All statistical analysis was performed using GraphPad Prism v6.0 (GraphPad Inc., La Jolla, CA, USA) software. All data were reported as the mean ± SD. The differences between two groups were analyzed using T-test and the differences among multiple groups were analyzed using one-way ANOVA or two-way ANOVA followed by Tukey’s test. Correlations between two groups were analyzed by the Person's Rank-Order method. Kaplan-Meier curve (Log-rank tests) was used to determine any significant associations of patient outcome and hypoxia score or NOX4. P<0.05 was considered statistically significant.