Patients and clinical samples
The study encompassed the procurement of plasma specimens from a cohort of sixty patients diagnosed with PDAC and thirty healthy individuals who underwent routine health examinations, all of whom were enrolled at the Affiliated Hospital of Nantong University between the months of December 2021 and June 2023(Supplementary Table 1). The research was sanctioned by the Ethics Committee of the Affiliated Hospital of Nantong University under the approval number 2021-K129-01, and all participants provided informed consent.
DNA extraction and Bisulfite Conversion
Peripheral blood samples of 10 mL placed in EDTA anticoagulant tubes, and refrigerated at 4℃ for two low-temperature centrifugations (1600×g and 16000×g) within 2 h. The samples were then immediately stored at -80℃. Fresh frozen tissue samples were stored at -80℃ immediately after surgery. Extract DNA from tissue samples using phenol-chloroform method, and extract cfDNA from plasma using the Blood Plasma cfDNA Extraction Kit (Qiagen, DP339). Store both samples at -20°C. Extract 500ng of DNA from both tissue and plasma samples and modify the DNA with sodium bisulfite according to the EZ DNA Methylation™ Kit (Zymo research, D5001) protocol. This process converts unmethylated cytosine to uracil, while 5-mC remains unchanged, allowing for the differentiation of methylated and unmethylated genes. The final elution volume for tissue samples is 30 µL, while for plasma samples, a volume of 10 µL is necessary to ensure sufficient DNA concentration. The DNA from normal male human brain tissue was fully methylated using M.SssI methyltransferase (Zymo research, D5011) as a positive control for human methylation. ddH2O was used as a negative control. The Bisulfite DNA (BS-DNA) was stored at -20°C.
Whole genome bisulfite sequencing (WGBS) library construction and data analysis
After satisfactory detection of plasma DNA from 3 cases of PDAC and 3 normal samples, a certain ratio of lambda DNA was added to serve as quality control for Bisulfite conversion. Genomic DNA was randomly fragmented to 200–400 bp using Covaris M220. The fragmented DNA was repaired, A-tailed, and connected with methylation-modified sequencing adapters. Subsequently, Bisulfite treatment occurred, followed by PCR amplification to obtain the final WGBS library. After library construction, we first used Qubit 2.0 for preliminary quantification, diluting the library to 1ng/µL. Then, insert fragment length was detected using Agilent 2100, and the effective concentration of the library was accurately quantified using Q-PCR (library effective concentration > 2nM) to ensure library quality. After library inspection, different libraries were pooled according to effective concentration and target downstream data volume requirements for Illumina HiSeq sequencing. After obtaining the raw sequencing reads, we conducted bioinformatic analysis.
Multiple Quantitative Methylation-Specific Polymerase Chain Reaction (M-QMSP) of Sample Target Fragment
Based on pre-existing differential methylation sites, determine the region containing differential methylation sites to be validated. The DNA fragment of this region is the target fragment. Design QMSP primers and probes using this sequence as a template to verify the methylation level differences between the case and control groups in this region. The primer design was completed using the methprimer1.0 online software, and Beacon Designer was used for probe design. Using β-actin (ACTβ) as an internal control gene, design primers and probes in a CpG-free region to avoid false negatives25, 26. Primers and probes were synthesized by Sangon Biotech (Shanghai). The QMSP total reaction system is 20 µL, including 0.4 µL of 10 µmol/L upstream and downstream primers, 0.2 µL of 10 µmol/L probe, 2 µL of BS-DNA, 10 µL of AceQ qPCR Probe Master Mix (Vazyme, Q112), 5 µL of ddH2O, cycling parameters: 95℃ for 10 min; 95℃ for 15 s, 58℃ for 1 min, a total of 55 cycles. Fluorescence signals were collected at 58℃ using the LightCycler® 96 Instrument. Each sample was repeated three times, and if no curve amplified twice, it was considered invalid. For each gene, the percent of methylated reference (PMR) of each sample was calculated using the following formula: PMR = 2−ΔΔCt × 100%, and ΔΔCt = [Ct(gene) - Ct(ACTβ)](Sample) - [Ct(gene) - Ct(ACTβ)](M.SssI treated normal DNA)25, 27(Fig. 1).
Cell culture, reagents, and transfection
HPDE6-C7, PANC-1, MIAPaCa-2, CFPAC-1, SW1990, BXPC-3, and AsPC-1 cell lines were purchased from the cell bank of Shanghai Academy of Biological Sciences, Chinese Academy of Sciences. The cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin streptomycin. The methods of plasmid transfection and lentivirus infection are described in the supplementary methods.
Methylation-Specific Polymerase Chain Reaction(MSP)
The bisulfite conversion of tissue or cellular DNA, followed by preparing the reaction mixture in the dark. The system is 10 µL, including 0.4 µL of 10µmol/L upstream and downstream primers, 2 µL of BS-DNA, 10 µL of SYBR Green Master Mix, 7.2 µL of ddH2O, cycling parameters: 95℃ for 10 min; 95℃ for 15 s, 60℃ for 30 s, 72℃ for 30 s, a total of 36 cycles.
RNA isolation and quantitative real-time PCR (qRT-PCR) analysis
Total RNA was isolated using Reagent (Vazyme, R401-01). cDNA was prepared using the
PureScript II 1st Strand cDNA Synthesis Kit (Vazyme, PR211-01). qRT-PCR analysis was
performed using LightCycler® 96 Instrument.
Agarose gel electrophoresis
Add 50 mL 1×TBE to 1g agarose, heat for 1 min in microwave, then mix with green dye. Pour the agarose added with nucleic acid dye into the inner groove of the rubber plate, insert the comb, and let it stand at room temperature for 30 min. Carefully pull out the comb after the gel solidifies. Add 1×TBE to tank, submerge gel near black pole, run at 120V for 30 min, then image.
Immunohistochemical (IHC)
The surgically excised PDAC tissue specimens were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. The tissue sections or fixed cells were treated with 3% hydrogen peroxide to block endogenous peroxidase activity. Antigen retrieval was performed, followed by blocking of nonspecific binding with 4% normal goat serum (Gibco). Subsequently, the tissue sections were incubated with the primary antibody, and then with goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) at a dilution of 1:1000. The sections were then stained using 3,3’-diaminobenzidine (DAB) as the chromogenic substrate solution.
Western blot
Proteins were meticulously extracted from cultured cells utilizing RIPA buffer (Beyotime) at a controlled temperature of 4°C. Subsequently, the extracted proteins were subjected to immunoblotting analysis, employing the corresponding antibodies in conjunction with a protease inhibitor cocktail and phosphatase inhibitor cocktail to ensure the integrity of the proteins. The concentration of the proteins was accurately determined using a BCA Protein Assay Kit (Beyotime). The proteins present in the cell lysates were then meticulously separated by SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (Sangon Biotech). These membranes were subsequently probed with the specified primary antibodies and HRP-conjugated secondary antibodies for further analysis.
Wound-Healing Assay
Cells are inoculated into a six-well plate, with 5x105 cells in each well, and incubated at 37°C in a humidified incubator with 5% CO2. Once the cells reach confluence, a straight edge is aligned, and using a 200 µL pipette tip, two perpendicular lines are drawn across the wells, marking the scratch. At 0 h, 12 h, and 24 h, photographs are taken under the microscope to document the healing progression of the scratch assay.
Clonogenic cell survival Assay
Cells were seeded into a six-well plate at a density of 700 cells per well. After two weeks of culture in complete medium, the cells were fixed with 4% paraformaldehyde and stained with 1mL of crystal violet solution for 15 min. The cells were then observed and photographed under a microscope.
Oil Red O staining
Cells were seeded in a six-well plate at a density of 5x105 cells per well. Following the experimental groups' assignment, either complete medium (basic medium + 10%FBS) or lipid medium (Oleic acid: Palmitic acid = 0.4 mM:0.2 mM) was added to each well. Incubate plates at 37°C, 5% CO2 for 48 h, then stain with Oil Red O kit (Beyotime, C0157S).
Statistics analysis
The findings are depicted as mean ± SEM. PMR calculations were executed in Excel 2021. Statistical assessments employed paired t-tests for tissue samples and unpaired t-tests for plasma samples, examining methylation status of both tumor patients and healthy individuals. One-way ANOVA was used to analyze the correlation between DNA methylation and clinical stage. GraphPad Prism 8.0 created scatter plots while SPSS v. 19.0 software drew ROC curves and conducted AUC evaluations. The Youden index = sensitivity + specificity − 1, with the maximum value in the dataset representing the optimal cutoff, indicating equal significance of sensitivity and specificity. P < 0.05 is considered statistically significant.