Bioinformatics analysis

Gene expression transcripts (RNA-seq) and clinical data from 179 PAAD and 4 paracancerous tissues were sourced from TCGA (https://docs.gdc.cancer.gov/), while 167 normal pancreatic gene transcripts were obtained from the GTEx database (https://gtexportal.org/home/). Additionally, microarray expression data from the GEO database (https://www.ncbi.nlm.nih.gov/geo/) included GSE62165, GSE15471, and GSE71989 datasets. Transcript differential analysis employed “limma,” “edgeR,” and “stats” for statistical analysis, and data visualization utilized the “ggplot2” software package. Survival analysis employed the “survival” package, with survival curves plotted using “survminer” and “ggplot2.” ROC analysis was conducted using the “pROC” package, and results were visualized using “ggplot2.”

Specimen collection

Ninety PC tissue and paracancerous specimens (1–2 cm of normal pancreatic tissue adjacent to tumor parenchyma) with clinicopathologic data were procured from patients at the Hepatobiliary Surgery Department of the Affiliated Hospital of Guizhou Medical University and the Affiliated Tumor Hospital, who underwent surgical resection from April 1, 2020, to April 1, 2023. Ethical approval for specimen collection and clinical data was granted by the Ethics Committee of Guizhou Medical University (2019 Ethical Review No. 073), and participating patients provided informed consent orally and through written agreements.

Cell culture

The normal human pancreatic ductal epithelial cells hTERT-HPNE and PC cell lines (AsPC-1, BxPC-3, PANC-1, MIA PaCa-2, SW1990) were procured from the Cell Bank of the Chinese Academy of Sciences. Culturing conditions included DMEM (Gibco, USA) with 10% FBS (Gemini, USA) and 1% penicillin for hTERT-HPNE, PANC-1, MIA PaCa-2, and SW1990, while AsPC-1 and BxPC-3 were cultured in RPMI 1640 (Gibco, USA) with 10% FBS and 1% penicillin. All cells were maintained in a 37 °C, 5% CO2, 95% air incubator (Thermo, USA).

Stabilized cell line and plasmid construction and transfection

Lentiviruses including negative control lentivirus Vector (Lv-NC), PELI1 overexpression lentivirus (Lv-PELI1), PELI1 knockdown lentiviruses (sh-NC, sh-PELI1#1, sh-PELI1#2), and Myc-RPS3 were obtained from Shanghai Genechem Co. Ubiquitin (HA-Ub) overexpression plasmids (HA-WT, HA-K6, K11, K27, K29, K33, K48, K63) were purchased from Shanghai Sangon Bioengineering Co. Plasmid transfections utilized lipofectamine 3000 (Invitrogen, USA) and Opti-MEM (Gibco, USA).

Antibodies and reagents

During the study, various antibodies were employed, including PELI1 (ab199336, Abcam) for Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), and immunoprecipitation (IP), Cyclin E1 (11554–1-AP, Proteintech), Cyclin D1 (60186–1-Ig, Proteintech), CDK7 (#2916, CST), CDK4 (GB111602–100, Servicebio), p27 (25614–1-AP, Proteintech), E-cadherin (20874–1-AP, Proteintech), N-cadherin (22018–1-AP, Proteintech), Vimentin (10366–1-AP, Proteintech), Snail (10399–1-AP, Proteintech), Alpha Tubulin (66031–1-Ig, Proteintech), RPS3 (66046–1-Ig for WB/IHC/IF, 11990–1-AP for IP, Proteintech), Ki67 (27309–1-AP, Proteintech), PCNA (10205–2-AP, Proteintech), p53 (66031–1-Ig for WB, 10442–1-AP for IP/mIHC, Proteintech), Flag (66008–4-Ig, Proteintech), HA (66006–2-Ig, Proteintech), Myc (60003–2-Ig, Proteintech). Fluorescent secondary antibodies included mouse secondary antibody (SA00009–1, Proteintech) and rabbit secondary antibody (SA00013–4, Proteintech).

Chemical reagents employed in the experiment consisted of CHX (Aladdin, China), MG132 (Sigma, USA), four-color multiplex immunohistochemistry kit (Absin, abs50012, China), PI (Solarbio, China), DAPI (Solarbio, China), and D-Luciferin Potassium salt (PerkinElmer, USA).

Immunohistochemistry and multiple fluorescence immunohistochemistry

Tissues were fixed, dehydrated, and paraffin-embedded with 4 μm thick sections. Sections were deparaffinized with xylene, hydrated with an ethanol gradient, and subjected to antigen repair using EDTA (Solarbio, China). Endogenous peroxidase was blocked with H2O2, followed by confinement with 5% BSA (Solarbio, China). Antibodies were applied overnight at 4 °C, followed by washing with PBST and incubation with secondary antibodies at room temperature for 2 hours. Sections were developed with diaminobenzidine working solution (ZSGB-Bio, China), dehydrated with an ethanol gradient, and transparently sectioned using xylene. Slides were sealed with neutral resin (Solarbio, China). IHC scoring was performed by two pathologists based on staining intensity and cell count.The intensity of cell staining was scored on a 4-point scale, (negative = 0, weakly positive = 1, positive = 2, strongly positive = 3), and the percentage of positive cells was scored on a 5-point scale, < 1% = 0, 1–25% = 1, 26–50% = 2, 51–75% = 3, and > 75% = 4. The two scores were then multiplied to give a final score. Scores below less than 7 were considered low expression, and scores greater than or equal to 7 were considered high expression.

For multiple fluorescence IHC, the same treatment as IHC staining was applied, with an additional incubation step using staining solution. After antibody staining, DAPI was applied for nuclei staining, and slides were sealed with an anti-fluorescence quencher. TBST washes were performed between each step, and fluorescence intensity was observed under a laser confocal microscope (Olympus, Japan).

Hematoxylin-eosin (HE) staining

Livers from mice in intrasplenic injection to construct a liver metastasis model were fixed, dehydrated, paraffin-embedded, and sectioned. Deparaffinization, hydration, and staining with hematoxylin and eosin were performed, followed by dehydration, xylene transparency, and blocking with neutral resin. Metastatic foci were counted through photography under an inverted microscope (Nikon, Japan).

RNA extraction and qRT-PCR experiments

TRIzol (Invitrogen, USA) reagent was used to extract total RNA from PC tissues and cells, and a NanoDrop spectrophotometer (Thermo, USA) was used to measure the concentration and mass of RNA with A260/A280 in the range of 1.8–2.0. RNA was reverse transcribed to cDNA using a PrimeScript RT kit (TaKaRa, Japan), and mRNA expression was analyzed using a Premix Ex TaqTM (Takara, Japan) kit and a real-time fluorescence quantitative PCR instrument. The Forward primer for PELI1 (5′- GAAATTGGTCCCCAGCCCTC-3′), Reverse primer (5′-GCGAGAGGGTGAAGTGTTGT-3′), Forward primer for α-Tubulin (5 ‘-ACCAACCTGGTGCCCTATCC-3’), Reverse primer (5′-CAAGCATTGGTGATCTCTCTGCTACA-3′). The Forward primer for RPS3 (5′-TACTACGTTGACACTGCTGTGCG-3′), Reverse primer (5′-GGTGGTGGGCAGTATCTCATCTT-3 ‘).

Western blot assay

Total protein was extracted from cells or tissues by adding a mixture of RIPA lysis buffer (Solarbio, China) and protease inhibitor (Boster, China) at 4 °C with centrifugation at 12,000 rpm for 15 min. The supernatant was collected, and protein quantification was performed using the BCA kit (Solarbio, China). Proteins were denatured at 95 °C, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to PVDF membranes (Meck, USA). After blocking with 5% skimmed milk powder at room temperature for 2 h, overnight incubation with primary antibodies at 4 °C, and subsequent incubation with secondary antibodies at room temperature for 2 h, membranes were washed with TBST containing 0.1% Tween (Solarbio, China). ECL chemiluminescent solution (Millipore, USA) was applied, and protein expression levels were analysed using the ChemiDocTM imager (Bio-Rad, USA).