2.1 PDAC patient cohort

A total of 128 patients with PDAC who underwent surgical resection in the Department of Visceral Surgery at the University Hospital Marburg were enrolled in our study (see Table 1). All tumors were histologically staged by an experienced pathologist according to UICC-TNM (Union for International Cancer Control; tumor, node, metastasis) classification 2017 [15]. Samples were obtained from the tumor bank of the Department of Pathology and Department of Visceral, Thoracic, and Vascular Surgery at Marburg University. Ethical approval was granted by the local ethics committee at Marburg University, Faculty of Medicine (File Nr. 5/03). All patients provided written consent prior to participating in this study.

Table 1 Clinical data on the PDAC cohort used in this study for NGS analyses (mRNA expression) and for immunohistochemistry (protein)2.2 Cell culture

Panc89 cells were cultured in RPMI-1640 (GibcoTM, Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma‒Aldrich, Dreieich, Germany), 0.1 mg/mL penicillin‒streptomycin (GibcoTM, Life Technologies) and 1 mM sodium pyruvate (GibcoTM, Life Technologies). Panc1, HPAC, CFPAC, PaTu8988T, S007, MiaPaCa and BxPC3 cells were cultured in DMEM (GibcoTM, Life Technologies) supplemented with 10% (v/v) FBS (Sigma‒Aldrich), 0.1 mg/mL penicillin‒streptomycin (GibcoTM, Life Technologies) and 1 mM sodium pyruvate (GibcoTM, Life Technologies). All cell lines were cultivated in a humidified incubator at 37 °C and 5% CO2, and routinely tested for mycoplasma contamination.

2.3 MiR-181a-5p mimic or inhibitor transfection

Lipofectamine™ RNAiMAX (Invitrogen) was used for transient transfection according to the manufacturer’s instructions. Cells were seeded in 6-well plates at a density of 500,000 cells/well and transfected with 0.01 μM hsa-miR-181a-5p mimic (miScript, Qiagen, Hilden, Germany) or inhibitor (miRCURY, Qiagen) after cell attachment. ON-TARGET plus nontargeting control pool (0.01 μM; Horizon Discovery, Cambridge, UK) was used as control miRNA. After 48 h, the cells were harvested, and miRNA expression was analyzed by RT‒qPCR. The sequence of the miR-181a-5p mimic was 5’-AACAUUCAACGCUGUCGGUGAGU’ 3’, and the sequence of the miR-181a-5p antisense (inhibitor) was 5’-ACTCACCGACAGCGTTGAATG’ 3’.

2.4 Transient transfection for NEAT1 knockdown

NEAT1 knockdown in cells was performed utilizing Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Cells were seeded in 6-well plates at a density of 400,000 cells/well and transfected with 0.02 μM silencer select NEAT1 siRNA (Thermo Fisher Scientific, Dreieich, Germany) after attachment. ON-TARGET plus nontargeting control pool (0.01 μM; Horizon Discovery, Cambridge, UK) was used as the control siRNA. After 24 h, cells were harvested, and lncRNA expression was analyzed by RT‒qPCR.

2.5 Transient transfection for NEAT1 overexpression

NEAT1 overexpression in cells was performed utilizing Lipofectamine LTX & PLUS Reagent (Invitrogen) according to the manufacturer’s instructions. Cells were seeded in 6-well plates at a density of 400,000 cells/well. The pcDNA3.1NEAT1 expression vector was cloned from a 3.7 kb NEAT1 fragment in pCRII_Topo (Addgene, USA) into the pcDNA3.1(-) vector by GenScript (Rijswijk, Netherlands). Cells were transfected with either 2.5 μg of pcDNA3.1-NEAT1 or with the pcDNA3.1 empty vector. After 48 h, cells were harvested, and lncRNA expression was analyzed by RT‒qPCR.

2.6 Reverse transcription-quantitative polymerase chain reaction (RT‒qPCR)

Total miRNA from cells was isolated using the miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A MiRCURY LNA RT Kit (Qiagen) was used for reverse transcription, and a miRCURY LNA SYBR® Green PCR Kit was used for quantifying miRNA expression according to the manufacturer’s instructions. The YP00203_U6 snRNA miRCURY LNA PCR Assay (Qiagen) and miRCURY miRNA Assay hsa-miR-181a-5p (Qiagen) were used for the quantification of relative miR-181a-5p expression. For total mRNA and lncRNA isolation, Qiazol (Qiagen) was used, and then 2 μg of isolated RNA was transcribed into cDNA using an EcoDryTM kit (Takara Bio, Inc.). RT‒qPCR was performed with iTaqTM Universal SYBR Green Supermix (Bio-Rad Laboratories GmbH, Göttingen, Germany). QuantiTect Primer Assay (Qiagen) of forward and reverse primers were diluted 1:10 in a total reaction volume of 20 μl using a StepOnePlus Real-time PCR system (ABI, Thermo Fisher Scientific, USA). The primers XS13 (ribosomal RPLP0 gene) (fw: 5’-TGG GCA AGA ACA CCA TGA TG-3’; rev: 5’-AGT TTC TCC AGA GCT GGG TTG T-3’) were used for internal normalization of gene expression, and relative gene expression was calculated by the 2-ΔΔCt method.

2.7 Protein extraction and Western blot

Cells were lysed using RIPA buffer (50 mM HEPES pH 7.4; 150 mM 542 NaCl; 1% (v/v) NP-40; 0.5% (w/v) Natriumdeoxycholate; 0.1% (w/v) SDS; 10 mM phenantrolin; 10 mM EDTA) supplemented with protease inhibitor-cocktail (A32955, Thermo Scientific, Waltham, MA, USA), phosphatase inhibitor mix (A32957, Thermo Scientific, USA), and incubated on ice for 30 min. After centrifugation at 12,000 × g for 15 min at 4 °C, the protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Then, proteins were boiled at 95 °C for 5 min. The extracted proteins were separated by SDS‒PAGE on 10% gels and then transferred to nitrocellulose membranes (GE Healthcare, Düsseldorf, Germany) using wet transfer. The membranes were blocked with 5% (w/v) milk powder (MP) in TBST for 1 h and subsequently incubated with the following primary antibodies at 4 °C overnight: anti-ADAM8 (PA5-47047, Thermo Fisher Scientific, 1:1000), anti-β-Tubulin (NB600-936, 1:2000; Novus Biological, Wiesbaden, Germany), anti-STAT3 (9139S, 1:1000; Cell Signaling Technology, Leiden, The Netherlands), and anti-pSTAT3 (Tyr705) (9145S, Cell Signaling Technology, 1:1000). After washing with TBST three times, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature (1:4000; Abcam, Cambridge, UK). The membranes were washed three times with TBST and signals were detected using the Chemiluminescent HRP Substrate, Western Bright Sirius (Advansta, Hessisch Oldendorf, Germany) and the ChemiDoc MP Imaging System (Bio-Rad Laboratories GmbH). Signals were quantified using Image J.

2.8 Cell proliferation assay

A total of 5000 cells were seeded in each well of a 96-well plate overnight for attachment. A CellTiter-Glo 3D cell viability assay (Promega, Walldorf, Germany) was performed to detect cell proliferation at 24, 48 and 72 h. Then, 50 μL of CellTiter-Glo 3D reagent was added to each well and mixed by shaking for 15 minutes. After incubation at room temperature in the dark for 15 min, luminescence was measured with a microplate reader (FLUOstar OPTIMA Microplate Reader, BMG Labtech, Offenburg, Germany).

2.9 Scratch assay

A total of 50,000 cells in 70 μL of medium containing 10% FBS (v/v) were seeded per well of a Culture-Insert Well (Ibidi GmbH, Gräfelfing, Germany) in a 24-well plate overnight for attachment. Cells were starved overnight with medium supplemented with only 1% FBS. The medium was exchanged by fresh medium supplemented with 10% FBS on the next day and inserts were removed to start the migration assay. Images of each gap were taken at 0 and 10 h. Images were analyzed by Image J software.

2.10 Cell invasion assay

Invasion assays were performed using ThinCertTM Cell Culture Inserts with 8 μm pore diameters (Greiner Bio-One GmbH, Frickenhausen, Germany). The upper side of the Thincert was coated with 50 μL of Culture UltiMatrix RGF BME (Bio-Techne GmbH, Wiesbaden, Germany), which was diluted 1:2 with cold medium (without FBS) before seeding the cells. After solidifying for 1 h at 37 °C, 25,000 cells in 50 μL of medium containing 0.5% (v/v) FBS were seeded on the lower side of the Thincert. The cells were cultivated in an incubator for 5 h for attachment. Then, 750 μL of medium supplemented with 0.5% FBS was added to each well, and 250 μL of medium supplemented with 20% FBS was added to overlay the matrix. After 24 h, 4% (w/v) PFA was used to fix the cells for 30 min, and 0.3% Triton was used to permeabilize the cells for 30 min. The cells were subsequently stained with Hoechst 33,342 dye overnight for quantification. The Z-stack of five random viewing fields were recorded. For quantification, cells on the lower part of the Thincert (noninvasive cells) and cells in the matrix (invasive cells) were counted.

2.11 Lumit immunoassay

A total of 25,000 cells were seeded per well in a 96-well plate overnight for attachment. After that, the medium was replaced with 40 μL of fresh cell medium. Next, 10 μL of lysis solution (Promega) was added to each well, followed by vigorous shaking of the plate for 20 min. Then, 50 μL of antibody mixture (anti-STAT3 and anti-pSTAT3, Cell Signaling Technology; Lagit anti-mouse antibody-LagBit, and Lumit anti-rabbit antibody SmBit, Promega) was added to each well, and the plate was gently shaken for 2 min. The plate was then incubated at room temperature for 90 min. Before luminescence was measured, 25 μL of Lumit detection reagent (Promega) was added and the plate was gently shaken for 2 min.

2.12 Dual‑luciferase reporter gene assay

DNA sequences from NEAT1 containing either wild-type or mutant miR-181a-5p binding sites were synthesized and cloned via XhoI and NotI into psiCHECK2 to generate the corresponding reporter constructs (Fig. 4G). Panc89 cells were cotransfected with 0.4 μg of reporter construct and 0.01 μM miR-181a-5p mimic or control RNA (siCtrl). After 48 h, the cells were harvested and analyzed using a dual luciferase assay (Promega) according to the manufacturer’s instructions. All transfection assays were carried out in triplicate.

2.13 NEAT1-protein interaction assay

Biotinylated NEAT1_1 or antisense NEAT1_1 was constructed by Biosense Bioscience Co., Ltd. (Guangzhou, China), and then incubated with cellular protein lysates from Panc89 cells. Then, 300 ml of streptavidin beads were added. Recovered proteins associated with NEAT1_1 or antisense NEAT1_1 were detected by western blotting.

2.14 RNA binding protein immunoprecipitation (RIP) assay

RIP experiments were performed using the Magna Nuclear RIP™ kit (Millipore). Briefly, 3 × 107 cells were prepared for lysis. Magnetic bead-antibody complexes for immunoprecipitation were prepared by using 10 μg of STAT3 antibody (Cell Signaling Technology). Then, RNA binding protein‒RNA complexes (RBPs) were immunoprecipitated according to the manufacturer’s instructions. Fold enrichment was calculated based on the 2-ΔΔCt method.

2.15 Subcellular fractionation

Cells were collected as described and lysed using cytosolic lysis buffer supplemented with 5% NP-40 and 2 mM vanadyl ribonucleoside complex (VRC). After centrifugation at 1000 × g, the pellet was used to extract the nuclear proteins whereas the supernatant was used for extraction of cytoplasmic proteins. The supernatant was centrifuged at 13,000 × g, 1/10 of the total cytoplasmic cell lysate was used for protein detection, and 9/10 of the total cytoplasmic cell lysate for RNA extraction. Next, the pellet was treated with nuclear extraction buffer with 2 mM VRC added and centrifuged at 13,000 × g for 30 min. About 1/10 of the total nuclear cell lysate was used for protein detection and 9/10 of the total nuclear cell lysate for RNA extraction to verify enrichment of nuclear (Histone H3) and cytoplasmic proteins (GAPDH). Furthermore, qPCR analysis for nuclear RNA was performed with primers for U6 spliceosomal nuclear RNA and cytoplasmic RNA was detected using primers for ß-Actin. Enrichment for these RNAs was at least 5000× fold calculating the ratios of cytoplasmic over nuclear RNA for ß-Actin and at least 700× fold for U6 when calculating the ratio of nuclear over cytoplasmic RNA.

2.16 Ubiquitination assay and bortezomib treatment

Ubiquitination assays were conducted using a Signal Seeker™ Ubiquitination Detection Kit (Cytoskeleton Inc., Denver, CO, USA). Briefly, cells were used from siRNA transfection experiments or were incubated with or without 1 ng/mL bortezomib for 16 hours prior to attachment and then harvested. Next, cell lysates were prepared using BlastRTM Rapid Lysate Prep provided by this kit. After that, the protein concentration was detected. 1 mg of total protein was used for immunoprecipitation using Ubiquitination Affinity Beads and incubated on a rotating platform at 4 °C for two hours. About 20 μl of the lysate was saved as western input lysate control. Then the beads were collected by centrifugation at 3–5000 × g and washed three times. After the final wash and complete removal of buffer supernatant, 30 μl of Bead Elution Buffer was added. The beads were resuspended and incubated at room temperature for five minutes. Next, the bead suspension was gently transferred to the spin column and centrifuged at 9–10,000 × g for one minute at room temperature. Both of input lysate and IP lysate were denatured at 95 °C for five minutes followed by western blot. Primary antibodies of STAT3 and pSTAT3 were used as described above. This ubiquitination assay kit detected both of mono- and poly-ubiquitinated protein, so after ubiquitination precipitation and western blot, the displayed bands were all representative for ubiquitinated STAT3.

2.17 Statistical analysis

For statistical analyses of normal distributed values, Student’s t test, for non-normal distribution, Mann-Whitney U test (two groups comparison) and for more than 2 group comparisons, a Kruskal-Wallis test was used. Data were considered not significant (p > 0.05, no asterisk), significant * (p < 0.05), highly significant ** (p < 0.01), or very highly significant *** (p < 0.001) and are expressed as the mean ± S.D.