Patients and clinical specimens
We collected 20 pairs of human pancreatic tissues and adjacent (located > 5 cm away from tumor) normal tissue samples were collected from patients with pancreatic ductal adenocarcinoma (PDAC) who went through primary surgical treatment at Zhongda Hospital (Nanjing, China) with written informed consent. All the clinical specimens were snapped-frozen and stored in liquid nitrogen. The ethical approbation was conferred from Committees for Ethical Review in China Pharmaceutical University (Nanjing, China). Pathological diagnosis was made complying with the histology of tumor specimens or biopsy as well as examined by experienced pathologists. The patients’ clinical features are listed in Additional file 1: Table S1. The study was in accordance with all relevant ethical regulations for human research volunteers, and all participants submitted written informed consent.
Pancreatic cancer cell lines and cell culture
The human pancreatic cancer cell lines (Panc-1, MIApaca-2, SW1990, and BXpc-3), and human pancreatic duct epithelial (HPDE) cells used in the study were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The HPDE, Panc-1 and BXpc-3 cells were grown in RPMI-1640 (Cat# 11875093, Gibco); the MIApaca-2 cells were grown in DMEM medium (Cat# 11054020, Gibco), and SW1990 cells were grown in L-15 medium (Cat# 11415064, Gibco). All these mediums were complemented with 10% FBS (Cat# 10099141C, Gibco, USA), 100 U/mL penicillin and 100 ng/mL streptomycin (Cat# C0222, Beyotime, China). The cells were maintained in a humidified chamber supplemented with 5% CO2 at 37 °C. The spheroid pancreatic cancer cells were cultured as described in our previous study [11] in DMEM: F12 (Cat# 11765054, Gibco) complemented with 1% FBS, 1% methylcellulose (1500cP, Cat# 9004–67-5, Sigma), 100 ng/ml bFGF (Cat# 500-P18, PeproTech), 20 ng/ml EGF (Cat# 100–47, PeproTech), 2% B27 supplement (Cat# 17504044, Gibco), 1% ITS (5 mg/L insulin, 5 mg/L transferrin, 5 μg/L sodium selenite; Cat# 41400045, Gibco), 100 µg/ml streptomycin (Cat# 15070063, Gibco) and 100 U/ml penicillin under 5% CO2 at 37 °C. The 10-day-old spheroids were collected for the following assays.
RNA isolation, reverse transcription, and quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from pancreatic cancer cells and sample tissues using TRIzol reagent (Cat# 15596018, Invitrogen) and treated with DNase I (Cat# M2181, Promega). Reverse transcription reaction was achieved through usage of PrimeScript™ RT reagent Kit (Cat# RR037A, Takara). Diluted cDNA was employed to conduct qRT-PCR analysis by SYBR Premix Ex Taq II Kit (Cat# DRR041A, Takara) according to the product instruction with the proper primers listed in Additional file 2: Table S2.
Flow cytometry and cell sorting
Cells were stained using different antibodies conforming to the product instructions, and the used antibodies are provided as Additional file 3: Table S3. A FACSCalibur (BD Immunocytometry Systems) was employed to detect labeled cells or they were sorting with FACS Aria III (BD).
Plasmid construction and cell transfection
Super-Fidelity DNA Polymerase (Cat# P501-d1, Vazyme) was used to amplify the cDNAs of CTD-3252C9.4, IFI6 and IRF1, which were cloned into the expression vector pcDNA3.1 (Cat# V79020, Invitrogen). All PCR products were verified by DNA sequencing. In Additional file 3: Table S3, primers used for plasmid construction are listed. Cells were transfected with the above plasmids or purchased lentivirus pLVX-CTD-3252C9.4 (General Biosystems) for overexpressing the corresponding genes, and pcDNA3.1 (pcDNA-Ctrl) or pLVX-Ctrl were used as control. The plasmid construct map can be found in Additional file 4: Fig. S1. For knocking down, cells were transfected with Smart Silencers (RiboBio) that contains 3 siRNAs, coincident with si-Ctrl as negative control.
Proliferation, migration and Invasion assay
Cell proliferation was assayed using Cell Counting Kit 8 (CCK-8) assay, plate colony assay and 5-Ethynyl-2'-deoxyuridine (EdU) assay. CCK-8 and EdU assays were performed using Cell Counting Kit 8 (Cat# CK04, Dojindo) and EdU Cell Proliferation Assay Kit (Cat# C10310-1, RiboBio) base on the kits’ instructions. Plate colony assay was performed as described before [14]. Cell migration and invasion abilities were assessed by transwell assays based on published method [15, 16].
Apoptosis assay
Annexin V/propidium iodide (PI) staining, TUNEL assay and detection of apoptosis-related proteins (cytochrome-C, Bcl-2, Bax, cytochrome-C, caspase-3, cleaved-caspase-3, caspase-9, cleaved-caspase-9) were applied to estimate cell apoptosis, Annexin V/ PI staining were performed by using Annexin V-FITC/PI Apoptosis Detection Kit (Cat# KGA101, KeyGen Biotech), One-Step TUNEL Apoptosis Assay Kit (Cat# C1089, Beyotime) and Western Blot analysis, respectively.
Mitochondrial membrane potential detection
The mitochondrial membrane potential (Δψm) was assessed by JC-1 method. Concisely, JC-1 (Cat# C2005, Beyotime) working solutions were used to wash and incubate the indicated cells and this lasted for 20 min at 37 °C in the darkness. The cells were washed using JC-1 wash buffer after incubation and observed by a fluorescence microscope. In healthy cells with a Δψm indicating membrane depolarization, JC-1 forms JC-1 aggregates and shows red fluorescence. Whereas in apoptotic cells, JC-1 remains monomeric and exhibits green fluorescence.
Xenograft assays in nude mice
We purchased BALB/c nude mice (5–6 weeks old, 18–20 g) from the Model Animal Research Center at Nanjing University (Nanjing, China). They were raised under specific pathogen-free (SPF) conditions at China Pharmaceutical University. All of the animal studies obeyed with Institutional Animal Care and Use Committee (IACUC) regulations. The subcutaneous xenograft mouse model was used to evaluate tumor growth. BALB/c nude mice were randomly divided into two groups (5 mice/group) and accepted subcutaneous injection of different treated Panc-1 cells (1 × 106 suspended in 0.2 mL PBS). The tumor volumes were calculated individually using the formula: length × width 2 /2. The mice were sacrificed 27 days after injection, they were injected with sodium pentobarbital (150 mg/kg body weight, Sigma) for euthanasia to minimize suffering and distress according to AVMA Guidelines for the Euthanasia of Animals. The xenograft tumors were harvested and measured. For in vivo metastasis experiments, the mice were randomly divided into two groups (5 mice/group), and different treated Panc-1 cells (1 × 106 suspended in 0.1 mL PBS) were injected into the tail vein of each mouse. Metastases were then examined by bioluminescence images (BLI) by an IVIS Spectrum Xenogen Imaging System (Xenogen) on day 3, 10, 20 and 30. After 30 days, mice were humanely sacrificed by euthanasia. Entire mice livers and lungs tissues were harvested, photographed, and embedded in paraffin for H&E staining and Immunohistochemistry (IHC). The operators and investigators were blinded to the group allocation for all animal experiments. All animal experiments got approvement from the Ethics Committee of China Pharmaceutical University (Permit Number: 2162326).
Western blot analysis
The proteins were quantified using Bradford Protein Assay Kit (Cat# P0006, Beyotime). Equal amount of denatured protein was separated using 10% SDS-PAGE gels according to the molecular weight. Then, they were transferred to polyvinylidene difluoride membranes, which were immunoblotted with primary antibodies and detected with horseradish peroxidase-conjugated anti-IgG. Antibodies involved in the western blotting are provided in Additional file 3: Table S3. After that, membranes were visualized using Immobilon Western Chemiluminescent HRP Substrate (Cat# WBKLS0050, Millipore) and Image Lab™ Software (Bio-Rad).
Luciferase reporter assays
The IFI6 promoter region -1337 ~ + 199 bp construct was amplified from genomic DNA of Panc-1 cells. The full length and mutated IFI6 promoter constructs were cloned into the pGL3-basic reporter gene vector and verified by sequencing. The 293 T cells were used for transfection. Following the manufacturer’s instruction, luciferase activities were measured using Dual Luciferase Reporter system (Promega).
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed using the EZ-ChIP kit (Cat# 17–371, Millipore) according to the instructions. Cells were treated with 1% formaldehyde for cross-link at 37℃ for 10 min. Then, they were sonicated to 300 to 500 bp on average cracking with sodium dodecyl sulfate (SDS) lysis buffer. After incubating with proper antibodies (anti-IRF1) and washing, cross-linked DNA released from the protein-DNA complex and the eluted DNA was further purified and detected by qRT-PCR. The sequences of primers used for ChIP-qPCR are listed in Additional file 2: Table S2.
RNA pull-down assay
The full length CTD-3252C9.4 sequence was PCR amplified using a T7-containing primer and then reversely transcribed by in vitro Transcription T7 Kit (Cat# 6140, Takara). The targeted RNA was Biotin-labeled with Pierce™ RNA 3’ End Desthiobiotinylation Kit (Cat# 26,103, Thermo). The cells were lysedand streptavidin magnetic beads were used to capture the biotin-labeled CTD-3252C9.4 probe. The biotinylated RNAs were incubated with the protein extract from cells using Pierce™ Magnetic RNA–Protein Pull-Down Kit (Cat# 20,164, Thermo). The protein samples were further detected by Western Blot analysis after the magnetic beads (Cat# G7281, Promega) were eluted. The extracted protein was utilized as a positive control and antisense RNA as a negative control.
Statistical analysis
Each experiment was performed thrice and data were shown as the mean ± SEM. Statistical analysis was performed using GraphPad Prism 8.0. Student’s t-test was used for two-group comparisons. Pearson correlation analysis was used for analyzing the correlation between groups. P < 0.05 was considered statistically significant.
