Primary PAAD cells (cat. no. GPC0158) derived from pathological tissue samples of human pancreatic cancer obtained by surgery were purchased from China Center for Type Culture Collection. The present study was approved by the ethical review committee of Suizhou Hospital affiliated to Hubei University of Medicine (Suizhou, China). The primary PAAD cells were cultured in a serum-free complete culture medium for human pancreatic cancer primary cells (cat. no. CM-H153; Procell Life Science & Technology Co., Ltd.) in an incubator with humidified atmosphere at 37°C with 5% CO₂. The cells were passaged at 1:2 ratio once every 3 days and were used in experiments within 10 passages upon arrival.

Overexpression of AK4P1 in primary PAAD cells was achieved by transfection with AK4P1 overexpression plasmids (pGPU6-eGFP-Puro) constructed by Shanghai GenePharma Co., Ltd. at a final concentration of 1 µg/ml. The empty vector was used as negative control. AK4 gene expression was suppressed in primary PAAD cells (cell density, 60–70%) by transfection with AK4-targeting siRNA (A09011; GenePharma) constructed by GenePharma at a final concentration of 50 nM. The siRNA sequences were as follows: AK4-siRNA: 5′-CCCAGAACTTTGGTCTCCAGCATCT-3′; and AK4-SiRNA: 5′-GCACCGAAGTTGGTGAGATGGCAAA-3′. The negative control without homology with the target gene sequence was purchased from GenePharma (A06001; GenePharma; the sequence is not available by the company). Lipofectamine^® 3000 (cat. no. L3000075; Invitrogen; Thermo Fisher Scientific, Inc.) was used as the transfection reagent for transient transfection at room temperature for 10–15 min following the manufacturer's protocol. After 24 h of transfection, further experiments with cells followed.

AK4 knockout in primary PAAD cells was achieved using a customized CRISPR/Cas9 gene knockout kit [cat. no. L00694; GenScript; single guide (sg)RNA was designed on the CRISPR Design Tool (http://crispr.mit.edu/); the pU6gRNACas9 plasmid (C11002; GenePharma) was used for transfection; sgRNA sequence, 5′-GGGATGCGCTCCCCGGCCAT-3′, targets the promoter region of the AK4 gene] constructed by GenScript following the manufacturer's protocol and was confirmed by the negative results of RT-qPCR and western blotting assays evaluating the transcription and protein expression levels of this gene, respectively (data are shown in Fig. S1).

The present study was approved by the ethical review committee of Suizhou Hospital affiliated to Hubei University of Medicine; written informed consent was provided by each participant or the legal representative/guardian of the participant. The transcriptomic and survival data of the patients in the Genomic Data Commons (GDC) and the Cancer Genome Atlas (TCGA)-PAAD dataset were downloaded from Xena platform (20). The data from the exorBase database (http://www.exorbase.org/exoRBase/download/toIndex) were downloaded to explore exosomal lncRNAs with significant differences in their abundances in circulating exosomes derived from patients with PAAD and from healthy volunteers. PAAD pathological tissue specimens and the paired adjacent non-cancerous tissue specimens (~2 cm from the tumoral tissue) were collected during surgery from 31 patients with PAAD who received radical surgery before adjuvant chemo/radiotherapy in our facility between January 2019 and June 2020. The inclusion criteria were as follows: i) Patients were 19–75 years old; ii) patients with PAAD were diagnosed by CT and MRI before surgery, and confirmed by pathological examinations after surgery; iii) Patients with PAAD had not received radiotherapy and chemotherapy before surgery; and iv) the basic information of the patients was complete. The exclusion criteria were as follows: i) Metastatic pancreatic cancer; ii) pancreatic tissue that had not been confirmed by pathological examination; and iii) the data were incomplete and difficult to be included in statistical analysis. The tissue specimens were stored in liquid nitrogen before analysis. Circulating exosomes were extracted from frozen plasma samples derived from each of the 31 PAAD patient or 25 healthy individuals who received routine medical check-ups at our facility by ultracentrifugation following a previously described procedure (21) and were verified by transmission electronic microscopy before cryopreservation (TEM image of the exosomes is shown in Fig. S2). In short, peripheral blood was centrifuged at 2,000 × g for 10 min at 4°C to collect plasma, and then centrifuged at 4,000 × g for 20 min at 4°C, 12,000 × g for 30 min at 4°C, filtered with a 0.22-µm filter to collect the plasma and centrifuged at 100,000 × g for 60 min at 4°C twice, and finally the pellet was resuspended in PBS for use in follow-up analysis. Exosomes (20 µl) were pipetted onto the copper net dropwise and left for 5 min at RT. The excess liquid was absorbed at the side using filter paper, then 30 µl of 2% phosphotungstic acid (cat. no. C0681090235; Nanjing Reagent) was added dropwise, and negative staining conducted for 3 min at RT. After the negative staining, the excess liquid was again absorbed with filter paper, and baking was performed under an incandescent lamp for 10 min. The shape and size of the exosomes was examined with a transmission electron microscope at 80 kV acceleration voltage. Clinicopathological characteristics of the patients are shown in Table SI. Paraffin-embedded specimens of 83 patients diagnosed with PAAD in Suizhou Hospital between March 2012 and July 2018 were collected. qPCR was performed to detect the expression levels of AK4 and AK4P1. These patients were followed up by telephone for survival analysis. The clinicopathological characteristics of these patients are presented in Table SII. The patients were divided into high and low groups based on the median of the transcription levels of AK4P1 and AK4 as detected by RT-qPCR in their tissue specimens and the overall survival of these patients was compared.

Total RNA from all samples was extracted by TRIzol^® method (Invitrogen; Thermo Fisher Scientific, Inc.). Primers were designed for the specific detection of AK4 or AK4P1 gene transcript and were constructed by GeneCopoeia, Inc. The primer sequences were as follows: AK4 forward, 5′-CTTGTGCCAGCTTCCCCGGCTC-3′ and reverse, 5′-CCTCGCGAAGGCAATGGCT-3′; AK4P1 forward, 5′-GTTGTCTTAAAAGTCTCTCCTTCCCCCTGT-3′ and reverse, 5′-CCTCGCGAAGGCAATGGCT-3′; and β-actin forward, 5′-CTCTTCCAGCCTTCCTTCCT-3′ and reverse, 5′-AGCACTGTGTTGGCGTACAG-3′. qPCR was performed using BlazeTaq One-Step SYBR Green RT-qPCR Kit (GeneCopoeia, Inc.) and a thermocycler using 1 µg cDNA as the template in each assay. The thermocycling conditions were as follows: Pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 sec and extension at 60°C for 30 sec (40 cycles); and melting curve: 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec). β-actin was used as housekeeping gene for the comparison of AK4P1 or AK4 gene transcription levels among tissue specimens or cells cultured in vitro and AK4P1 transcription levels in the exosome samples. Data were analyzed by the 2^−∆∆Cq method (22).

Protein levels of AK4 in the tissue specimens or cells cultured in vitro were assessed by western blotting using β-actin as internal reference gene. In brief, total protein from tissue specimens or cells cultured in vitro was extracted using harsh RIPA lysis buffer (cat. no. 89900; Thermo Fisher Scientific, Inc.). The protein concentration was determined using a BCA protein concentration assay kit (cat. no. 23227; Thermo Fisher Scientific, Inc.). A total of 30 µg protein was used in each western blot assay. The protein sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and the protein was transferred to a PVDF membrane (cat. no. ab133411; Abcam). The PVDF membrane was incubated for 2 h in a blocking solution (TBS with 0.1% Tween-20 solution containing 5% BSA (cat. no. A8010; Beijing Solarbio Science & Technology Co., Ltd.; pH 7.4) at room temperature. Then, the PVDF membrane was incubated with antibody at 4°C overnight. The membrane was then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:3,000; cat. no. ab6721; Abcam) at room temperature for 3 h. Digital imaging and signal quantification were performed using the ECL Plus Western Blotting Substrate (cat. no. 32134; Thermo Fisher Scientific, Inc.) using Bio-Rad Image Lab Software (version 4.0; Bio-Rad Laboratories, Inc.). Primary antibodies used in western blot assays were as follows: Anti-AK4 mouse monoclonal antibody (dilution, 1:1,000; cat. no. sc-271161; Santa Cruz Biotechnology, Inc.) and anti-β-actin rabbit monoclonal antibody (dilution, 1:1,000; cat. no. ab115777; Abcam).

PAAD cell viability was evaluated by Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.). Briefly, the cells were inoculated on a 96-well-plate at an initial density of 5×10³ cells/well and were cultured in serum-free complete culture medium for human pancreatic cancer primary cells at 37°C for 48 h; after which, the cells were incubated with 10 µl CCK-8 working solution at 37°C for 1 h. Absorbance was measured at an optical density of 450 nm using a microplate reader.

Apoptosis of the PAAD cells in response to gemcitabine challenge was evaluated using TUNEL method. The cells were inoculated on a 96-well-plate at a density of 2×10⁴ cells/well, before they were treated with gemcitabine (LY-188011; Abmole Bioscience Inc.) at a final concentration of 10 nM for 24 h. Apoptosis was assessed by TUNEL staining using a One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol and fluorimetry using a fluorescence microscope (excitation wavelength, 450–500 nm; emission wavelength, 515–565 nm). Briefly, the cells were washed with PBS, fixed with immunostaining fixative (P0098; Beyotime Institute of Biotechnology) for 30–60 min and washed with PBS, and PBS containing 0.1% Triton X-100 was added. The cells were incubated in an ice bath for 2 min. After washing with PBS, 50 µl TUNEL detection solution was added to the cells. The cells were incubated for 60 min in the dark at 37°C. Subsequently, the section was counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). After washing with PBS, the cells were re-suspended with 250–500 µl PBS and detected by fluorescence microscope (Olympus) at an objective magnification of ×800, with filter blocks for DAPI and fluorescein. At least 300 cells in each group were randomly selected for quantification, and the apoptosis rate was evaluated by the number of TUNEL staining positive cells.

GraphPad Prism software (version 9.02; GraphPad Software, Inc.) was used for statistical analysis. All data were normalized to the mean value of the data in the control group under the same experimental settings and are presented as the mean ± SD, unless otherwise indicated. An unpaired Student's t-test was used for comparing two groups of data. One-way analysis of variance was used for multi-group comparison followed by Tukey's post hoc test. Association analysis was performed by simple linear regression. Kaplan-Meier survival curve and log-rank test was used for survival analysis. A difference was considered statistically significant when P<0.05.

AK4P1 was identified as an exosomal lncRNA whose abundance in the circulating exosomes derived from patients with PAAD was significantly higher compared with that in the circulating exosomes derived from healthy individuals (Fig. 1A). It was also found that the abundance of AK4 mRNA in the circulating exosomes derived from patients with PAAD was significantly higher compare with that in the circulating exosomes derived from healthy individuals (Fig. 1B). To further explore the expression levels of AK4P1 and AK4 mRNA in publicly available data, the transcriptomic and survival data of patients in the GDC TCGA-PAAD dataset were downloaded from the Xena platform (Fig. 1C and D). The high transcription levels of either AK4P1 or AK4 mRNA were significantly associated with the decrease in the overall survival and tumor progression of patients with PAAD (Fig. 1E-H). The transcription levels of AK4P1 were significantly associated with those of AK4 mRNA in the tissue specimens of 179 patients with PAAD (Fig. 1I).

The expression levels of AK4 gene or AK4P1 pseudogene in 31 pairs of PAAD pathologic tissue specimens and para-carcinoma tissue specimens were compared. RT-qPCR results demonstrated that the transcription levels of AK4 and AK4P1 were significantly higher in PAAD tissue specimens compared with those in the para-carcinoma tissue specimens (Fig. 2A and B, respectively). Western blot results also showed a significant increase in AK4 protein expression levels in the PAAD tissue specimens compared with those in the para-carcinoma tissue specimens (Fig. 2C and D).

The expression levels of AK4 mRNA or AK4P1 pseudogene were further divided into high and low groups based on the median value, and it was observed that the mRNA expression levels of AK4 significantly associated with the transcription levels of lncRNA AK4P1 in the 31 PAAD tissue specimens (Fig. 2E). In addition, it was found that the exosomes obtained from patients with PAAD contained significantly higher levels of AK4P1 transcripts compared with those in the circulating exosomes derived from healthy individuals, as revealed by RT-qPCR (Fig. 2F). By integrating the AK4 and AK4P1 transcription levels in cryopreserved PAAD tissue specimens and the survival of each patient, it was identified that high expression levels of AK4 and AK4P1 in PAAD tissue specimens were significantly associated with decreased overall survival of patients (Fig. 2G and H).

To investigate the function of AK4P1 pseudogene in PAAD cells, the cells were transfected with AK4P1 overexpression vectors (Fig. 3A). It was observed that AK4P1 overexpression significantly upregulated the mRNA and protein levels of AK4 in PAAD cells in vitro (Fig. 3B and C, respectively). It was also found that AK4P1 overexpression significantly increased PAAD cell viability in vitro (Fig. 3D), and this upregulation was weakened by siRNA-mediated AK4 gene knockdown or abrogated by AK4 gene knockout achieved using CRISPR/Cas9 machinery. The cell apoptosis rate was normalized to the PAAD cells treated with gemcitabine (which could induce tumor cell apoptosis), and the cell apoptosis rate of different groups was compared (Fig. 3E), showing that AK4P1 overexpression significantly rescued PAAD cells from gemcitabine-induced cell apoptosis. In addition, when AK4P was overexpressed, the increase in apoptosis caused by AK4-KD was rescued, compared with the PAAD cells with siRNA-mediated AK4 gene knockdown. However, when AK4 gene was completely knocked out in PAAD cells, the increase in apoptosis level could not be rescued by AK4P1 overexpression (Fig. 3E). The representative TUNEL staining images are shown in Fig. S3.

Since the upregulation in AK4P1 levels in the circulating exosomes of patients with PAAD compared was confirmed, it was hypothesized that pancreatic cancer cells with high AK4P1 expression may deliver AK4P1 transcripts through exosomes, thereby affecting recipient cell growth and apoptosis. To investigate this, exosomes in the cell culture media conditioned by PAAD cells with or without AK4P1 overexpression were collected and used to incubate wild-type PAAD cells. RT-qPCR and western blot results showed that treatment with exosomes derived from AK4P1-overexpressing PAAD cells significantly increased the mRNA and protein expression levels of AK4 in the recipient PAAD cells compared with those treated with exosomes derived from donor cells without AK4P1 overexpression (Fig. 4A and B). It was further demonstrated that treatment with AK4P1-overexpressing PAAD cell-derived exosomes resulted in a significant increase in cell viability and a significant decrease in gemcitabine-induced cell apoptosis in the recipient cells compared with the recipients treated with exosomes derived from donor cells without AK4P1 overexpression (Fig. 4C and D). The representative TUNEL staining images are shown in Fig. S4.