2.1 Cells, reagents and antibodies

Cell Bank of the Chinese Academy of Sciences provided us the normal liver epithelial cell line (LO2), HCC cell lines (Huh7, LM3, and HepG2), and human monocytic leukemia cell line THP-1. Another human monocyte U937 was available from Procell Life Science & Technology (Wuhan, China). From Invitrogen (Carlsbad, CA, USA), DMEM, RPMI 1640 medium, FBS, and 1% penicillin/streptomycin were available. ApexBio Technology (Houston, TX, USA) provided AG-490. Human IL-4 and IL-13 recombinant protein were acquired from Gibco (Grand Island, NY, USA). ExoQuick Exosome Precipitation Kit was obtained from System Biosciences (Mountain View, CA, USA), and GM™ Exosome Isolation Reagent kit was from Geneseed Biology (Guangzhou, China). Ribo Biotech (Guangzhou, China) synthesized antisense oligonucleotide (ASO) targeting SNORD52 (SNORD52 ASO-1/-2) and negative control (ASO-NC), as well as pcDNA3.1-SNORD52 (OE-SNORD52) and OE-NC. Thermo Fisher Scientific (Waltham, MA, USA) provided transfection reagent Lipofectamine 3000. From Sigma‒Aldrich (San Luis, MO, USA), PKH26 Fluorescent Cell Linker Kit and PMA were purchased. Hoechst 33342 dye, exosome-positive marker CD9, and exosome-negative marker Calnexin were obtained from Cell Signaling Technology (Danvers, MA, USA). We acquired RIPA lysis buffer and BCA Protein Assay Kit from Beyotime Biotechnology (Shanghai, China). ECL Kit was obtained from Vazyme (Nanjing, China). RNA extraction Kit was acquired from Feijie Biotechnology (Shanghai, China). ReverTra Ace qPCR RT Kit was provided by TOYOBO Life Science (Shanghai, China). From novoprotein (Suzhou, China), SYBR High-Sensitivity qPCR Supermix was available. Phycoerythrin (PE)-conjugated anti-CD206 and fluorescein isothiocyanate (FITC)-conjugated anti-CD163 were obtained from Proteintech (Manchester, UK). Meanwhile, the primary antibodies Arginase-1, CD163, and STAT6 used in Western blotting were also purchased from Proteintech, while other primary antibodies including JAK2, p-JAK2, p-STAT6, exosome-positive markers (CD81 and TSG101), and the corresponding HRP-conjugated secondary antibodies were available from Abcam (Cambridge, UK).

2.2 Ethical statement

The present research study adheres to the principles outlined in the Declaration of Helsinki and has obtained ethical approval from the ethics committee at Taizhou Central Hospital (Taizhou University Hospital) (approval ID: 2020-SC-005). Informed consent was obtained from all participants after a comprehensive explanation of the study's objectives, procedures, potential risks, and benefits.

2.3 Patient samples

Five cases of patients diagnosed with HCC through pathological examination were included in the study at our hospital. These patients had not undergone any cancer treatments before being admitted. Additionally, three healthy individuals were selected as control subjects. Peripheral blood samples were collected from both HCC patients and healthy individuals, and then centrifuged at 3000 × g for 10 min at 4 °C to obtain plasma supernatant. The plasma samples were then centrifuged at a speed of 16,000 × g for 10 min at a temperature of 4 °C and kept in storage at − 80 °C until they were required.

2.4 Cell culture and treatment

Huh7, LM3, and HepG2 cells were grown in DMEM with an added 10% FBS and 1% penicillin/streptomycin, under a controlled atmosphere of 5% CO2 at 37 °C. When cultured THP-1 and U937 cells, the culture media were replaced with RPMI 1640 medium. PMA (50 nM) was used to induce THP-1 and U937 cells for differentiation into macrophage-like cells. M2 macrophage polarization was induced by addition of human IL-4 (20 ng/mL) and IL-13 (20 ng/mL) recombinant protein for 48 h.

2.5 Cell transfection

Following treatment with PMA for 24 h, THP-1 and U937 macrophages as well as Huh7 and HepG2 cells were transfected with OE-SNORD52 or OE-NC using Lipofectamine 3000 for 48 h, while the IL-4 and IL-13 treated THP-1/U937 macrophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h. The macrophages were then harvested for subsequent experiments.

2.6 Exosome isolation and characterization

HCC cell lines were cultured in DMEM with 10% FBS, without pre-existing exosomes, at 37 °C with 5% CO2. After 72 h of culture, the supernatant containing exosomes and cellular debris was separated by centrifugation. The collected supernatant was then processed for exosome extraction using a GM™ Exosome Isolation Reagent kit. The resulting mixture was incubated at 4 °C for 30 min before being centrifuged at a speed of 2000 × g for another 30 min. In addition, following the instructions of the ExoQuick Exosome Precipitation Kit, exosomes from plasma samples of HCC patients (HCC-Exo) or healthy individuals (control-Exo) were obtained. Finally, the extracted exosomes were resuspended in PBS for subsequent analysis.

Transmission electron microscopy (TEM; Hitachi, Tokyo, Japan) was employed to visualize the exosome microstructure. Nanoparticle tracking analysis (NTA) was utilized to assess the size distribution and concentration of the exosomes that were isolated. The presence of exosomal markers such as Calnexin, CD9, CD81, and TSG101 was determined through Western blotting.

2.7 Exosome internalization

Based on the guidelines from the PKH26 Fluorescent Cell Linker Kit, Huh7 cells-derived exosomes (Huh7-Exo) or HepG2 cells-derived exosomes (HepG2-Exo) were stained with PKH26 dye at 37 °C for a duration of 30 min. Afterward, the exosomes with labels were incubated with THP-1 macrophages for 6 h. After that, the cells were treated with 4% paraformaldehyde and fixed for 30 min. The nuclei were subsequently treated with Hoechst 33342 for a duration of 10 min. Subsequently, the internalization of exosomes was quantified using a confocal microscopy (Olympus, Tokyo, Japan).

2.8 Construction of a co-culture system

Huh7 cells alone and Huh7 cells that transfected with OE-SNORD52 as well as HepG2 cells alone and HepG2 cells that transfected with OE-SNORD52 were served as the donor cells to culture in a Transwell polyester permeable support. Meanwhile, THP-1 macrophages were served as receptor cells, which seeded on the lower chamber of Transwell culture plate. The cell density of each well is 2 × 105 cells. After an overnight incubation, the receptor THP-1 macrophages were collected for further experiments.

2.9 qRT-PCR

An RNA extraction Kit was applied for the extraction of the total RNA. The isolated RNA was used to produce cDNA via the ReverTra Ace qPCR RT Kit, followed by carrying out PCR analysis with the aid of a SYBR High-Sensitivity qPCR Supermix. The gene expression was determined by applying the 2−ΔΔCt approach, and GAPDH was used for normalization. Table 1 lists all the primers used.

Table 1 Real-time PCR Primer synthesis list2.10 Western blotting analysis

The RIPA buffer containing protease inhibitors was used for protein extraction. A total of 30 µg of extracted protein was electrophoresed on a 10% SDS-PAGE gel and transferred onto a PVDF membrane. The membranes were blocked with 5% nonfat milk, then incubated with primary antibodies including TSG101 (1:1500), CD81 (1:1500), CD9 (1:1500), Calnexin (1:1500), Arginase-1 (1:1000), CD163 (1:1000), JAK2 (1:1000), p-JAK2 (1:1000), p-STAT6 (1:1000), STAT6 (1:1000) and GAPDH (1:1000) at 4 °C for 12 h. Subsequently, HRP-conjugated secondary antibodies were added to the membrane and incubated for an additional hour. GAPDH was used as the internal reference. Finally, an ECL detection Kit was employed to visualize the bands more effectively using Gel-Pro Analyzer 4.0.

2.11 Flow cytometry analysis

THP-1 and/or U937 macrophages (5 × 105) were re-suspended in 500 μL binding buffer, followed by incubation with PE-conjugated anti-CD206 and FITC-conjugated anti-CD163 at 4 °C for 30 min in the dark. CD206-positive or CD163-positive macrophages were assessed using a FACScan flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

2.12 Statistical analysis

The data was analyzed using SPSS Statistics V22.0 and the results were presented as mean ± SD. Student’s t-test was used to compare differences between two groups, while one-way ANOVA with Tukey’s post-hoc test was employed for comparisons among multiple groups. A significance level of P < 0.05 was considered statistically significant.