Cell lines and cell culture
SW1990 cells were obtained from ATCC (USA). Cell lines were cultured in DMEM containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C with 5% CO2 humidification.
miR-193b-3p mimic (5ʹ-AACUGGCCCUCAAAGUCCCGCU-3ʹ), miR-193b-3p inhibitor (5ʹ-AGCGGGACUUUGAGGGCCAGUU-3ʹ), and negative control (NC, 5ʹ-CACUACUAUUGUGUAGAACAC-3ʹ) were purchased from Beyotime (Suzhou). The control pcDNA3.1 plasmid, TRIM62 overexpression plasmid, and c-Myc overexpression plasmid (Clontech, USA) were generated by Generay Technologies (Shanghai, China). Small interfering RNA (siRNA) targeting TRIM62 (sense, 5ʹ-GCAAGCUCUGCUCUUACUUTT-3ʹ; antisense 5ʹ-AAGUAAGAGCAGAGCUUGCTT-3ʹ) was obtained from Shanghai GenePharma Co., Ltd. Transfection was performed using Lipo2000 (Invitrogen, USA). Empty vector and nonspecific siRNA (siNC) were used as negative controls.
The control pShuttle-CMV adenovirus, TRIM62 overexpression adenovirus, and c-Myc overexpression adenovirus were obtained from Obio (Shanghai). Transfection was performed using Lipo2000 (Invitrogen, USA) according to the manufacturer’s instructions. At 48 h after transfection, the cell culture medium containing the viral particles was collected to infect SW1990 cells.
Macrophage polarization and cell co-culture
THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% exosome-free FBS. The medium was changed every 48 h. THP-1 cells were induced to differentiate into M0 macrophages by treatment with 100 ng/mL of phorbol myristic acetate (PMA, Sigma) for 24 h. To induce the M2 polarization of macrophages, 30 ng/mL of IL-4 (R&D Systems Inc., USA) was used to treat M0 macrophages for 24 h.
The conditioned medium (CM) of M0 or M2 macrophages was used for co-culture. To exclude the interference of exosomes in FBS, 10% (v/v) exosome-free FBS (SunBio, China) was used to configure the medium for the culture of macrophages. After 48 h, the supernatant of macrophages was collected and centrifuged at 5000 rpm for 5 min to remove excess cells. Finally, it was mixed at 1:1 with a complete medium containing 10% (v/v) exosome-free FBS and added to SW1990 cells for 24 h as the CM. Moreover, GW4869 (Umibio, China) was used as an exosome inhibitor, and macrophages were pretreated with 20 µM GW4869 for 24 h to inhibit the generation and secretion of exosomes.
Briefly, the supernatant medium from the macrophage culture was centrifuged at 500g for 15 min, 3000g for 15 min, and 12,000g for 30 min at 4 h. Exosomes were centrifuged at 140,000g for 80 min. After resuspension, they were centrifuged at 140,000g for 80 min. Then, the exosomes were placed on the copper grid for examination by transmission electron microscopy (TEM). The exosomes were labeled with a PKH-67 kit (Sigma, St. Louis, MO, USA) to detect their phagocytosis by SW1990 cells. SW1990 cells were treated with 100 µg/mL of M0 or M2 macrophage-derived exosomes.
Cell proliferation was determined using EdU assays. After the abovementioned treatment, cells were seeded into 24-well plates, incubated with fresh media containing 50 nM EdU reagent (RiboBioInc, China) for 2 h at 37 °C, fixed with 4% paraformaldehyde solution, followed by DAPI staining, and then subjected to fluorescence microscopy (Olympus, Tokyo, Japan).
Wound healing assay
The migration abilities of SW1990 cells were evaluated using wound healing assays. After the abovementioned treatment, SW1990 cells were seeded in a 35-mm culture dish (8 × 105 cells/dish). Wounds were created using a sterile pipette tip when the cells were fully confluent. After washing with phosphate-buffered saline (PBS) to clean the exfoliated cells, the cells were cultured in a pure medium without FBS. The images of wounds at the same position were taken at 0, 24, and 48 h to calculate the distance.
Transwell invasion assay
After the abovementioned treatment, transwell chambers were used to examine cell invasion. A culture medium containing 10% FBS was added to the lower chamber. Then, 5 × 104 SW1990 cells were suspended in serum-free culture medium and inoculated in the matrigel-coated upper chamber (BD Biosciences, USA). After 24 h of incubation, the SW1990 cells were removed from the upper chamber with a cotton swab. The cells that penetrated and adhered to the bottom of the filter membrane were fixed with 4% paraformaldehyde in PBS for 10 min, stained with 0.5% crystal violet for 20 min, and then imaged under a microscope.
Glutamine uptake assay
After the abovementioned treatment, a glutamine assay kit (ab197011; Abcam, USA) was used to determine the concentration of glutamine according to the manufacturer’s protocol. Based on the principle of glutamine conversion into glutamic acid and ammonia, the amount of glutamine was calculated by measuring the amount of ammonia.
Luciferase activity assay
SW1990 cells were transfected with an miR-193b-3p mimic/inhibitor. Then, the wild-type pGL3-promoter TRIM62 3ʹUTR (WT) or the mutant-type pGL3-promoter TRIM62 3ʹUTR (MUT) luciferase plasmid was transfected into SW1990 cells. The pRL-TK vector was transfected into SW1990 cells, which served as an internal control reporter. A dual-luciferase assay was performed according to the manufacturer’s protocol. The luciferase activity was evaluated using a Dual-Luciferase Reporter Assay system (Promega Biotech Co, Ltd, Beijing, China) at 48 h posttransfection and normalized to Renilla luciferase activity.
Quantitative RT-PCR (qRT-PCR)
RNAs were isolated using TRIzol and reverse-transcribed using Superscript II (Invitrogen, Shanghai). SYBR master mix (Bio-Rad, Philadelphia, PA) was used for qRT-PCR that was performed using the primers listed in Additional file 1: Table S1. The fold change for the relative gene expression was determined using the 2−ΔΔCt method. GAPDH/U6 served as an internal reference gene.
Cells were washed separately with ice-cold PBS, gently scraped with a cell spatula, and lysed using the radioimmunoprecipitation assay (RIPA) cell lysis buffer (Beyotime, Shanghai, China). Quantification was performed using the BCA protein concentration assay kit (Beyotime, Shanghai, China). Total protein samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking in 5% nonfat milk, the membranes were probed with antibodies against TRIM62 (ab102012; Abcam), c-Myc (ab32072), TSG101 (ab125011), CD9 (ab92726), CD63 (ab216130), or GAPDH (#5174; CST). The membranes were washed, probed with secondary antibodies (A0208, A0216; Beyotime, Shanghai, China), and then visualized using an ECL kit (Bio-Rad). Results were analyzed using the Image-Pro6.0 software.
Cycloheximide (CHX, 0.1 mg/ml; Sigma, Shanghai) was used to inhibit protein synthesis. SW1990 cells were treated with CHX for 0, 1, 4, and 8 h, cell lysates were obtained as described earlier, and then protein expression was assayed by western blotting. The CHX treatment time was plotted on the abscissa, and the amount of protein was plotted on the ordinate. The time required to degrade half the protein was the protein half-life.
Co-immunoprecipitation (Co-IP) and ubiquitination assay
Cell lysates were extracted using a RIPA buffer. The lysates were centrifuged at 12,000×g for 10 min at 4 °C. Total cell lysates were used for immunoprecipitation with anti-TRIM62 (ab154635; Abcam), anti-c-Myc (ab168727; Abcam), or normal IgG antibodies (sc-2027; Santa Cruz Biotechnology, Inc.), followed by protein A/G PLUS-Agarose beads (sc-2003; Santa Cruz Biotechnology, Inc.), at 4 °C for 2 h. Immunocomplexes were washed three times in the lysis buffer and subjected to western blotting using anti-TRIM62 (ab102012; Abcam), anti-c-Myc (ab32072; Abcam), or anti-Ubiquitin antibodies (ab7780; Abcam).
Experiments were performed according to the principles of the Committee on Ethics of Animal Experiments of Fudan University Shanghai Cancer Center. In a mice lung metastasis model, 5 × 106 SW1990 cells were inoculated through tail veins (n = 5). Exosomes (10 µg) derived from M0 macrophages (M0-exo) or M2 macrophages transfected with the miR-193b-3p inhibitor (M2/Inhibitor-exo) or NC (M2/NC-exo) were inoculated every 3 days. Otherwise, 5 × 106 SW1990 cells with TRIM62 and c-Myc overexpression adenovirus infection were inoculated through tail veins (n = 5). Mice were euthanized 6 weeks post injection, and the lung was examined and collected for hematoxylin–eosin (HE) staining. The number of lung nodules was recorded.
Two cohorts of PC cases in Fudan University Shanghai Cancer Center recruited from October 2019 to March 2021 were included in this study. Cohort 1 consisted of patients with tumor and adjacent normal tissues (n = 20), and cohort 2 consisted of patients with tumor (n = 60) and adjacent normal (n = 10) tissues. This study was approved by the Ethics Committee of Fudan University Shanghai Cancer Center and was performed according to the Declaration of Helsinki. Written informed consents were obtained.
Paraffin-embedded sections of 60 tumor tissues from cohort 2 were prepared for IHC staining. The tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and subjected to standard dewaxing and rehydration. The sections were incubated in citric acid buffer (pH 6.0) for 15 min for antigen retrieval, followed by incubation for 10 min with 3% H2O2 solution to inactivate endogenous enzymatic activities. Then, the sections were incubated with anti-TRIM62 (ab154635; Abcam) and anti-c-Myc (ab32072; Abcam) antibodies for 1 h at 25 °C, followed by HRP-conjugated anti-IgG antibody for 30 min at 20 °C. After washing three times with PBS, the peroxidase activity was visualized using 3,3ʹ-diaminobenzidine (OriGene Technologies, Inc., Rockville, MD, USA) at 25 °C for 10 s. Then, the sections were counterstained with hematoxylin (OriGene Technologies, Inc.) at room temperature for 3 min. Immunoreactivity was scored using the H-score system by two investigators based on the percentage of positive cells (0–4: 0, < 5%; 1, 5–25%; 2, 25–50%; 3, 50–75%; and 4, > 75%) and staining intensity (0–3: 0, negative; 1, weak; 2, moderate; and 3, strong), which ranged from 0 to 12. Based on the immunoreactivity scores, the patients were categorized into low-expression (H-score < 6) or high-expression (H-score ≥ 6) groups.
Data were analyzed using GraphPad8.4.3 (La Jolla, CA). Results are expressed as mean ± SD. Between-group differences were evaluated using Student’s t-test or ANOVA. The Kaplan–Meier method and log-rank tests were used to analyze and compare overall survival. P values of < 0.05 were considered to indicate statistical significance.