Normal human mammary epithelial cells MCF-10A (cat. no. MZ-0695), and breast cancer cells MCF-7 (cat. no. MZ-0113), BT-549 (cat. no. MZ-0031) and MDA-MB-231 (cat. no. 228845) were obtained from Ningbo Mingzhou Biotechnology Co., Ltd. Human umbilical vein endothelial cells (HUVECs; cat. no. CL-0122) were provided by PromoCell GmbH and passaged in endothelial cell growth medium (PromoCell GmbH). MCF-10A cells were cultured in mammary epithelium growth medium (Shanghai Yaji Biological Technology Co., Ltd.) with 100 ng/ml cholera toxin and 1% Penicillin/Streptomycin. BT-549 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) with 0.023 U/ml insulin and 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). MCF-7 and MDA-MB-231 cells were cultured in DMEM with 10% FBS, 50 IU/ml penicillin and 50 µg/ml streptomycin. Human monocytic leukemia THP-1 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences and cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). All cells were maintained at 37˚C in humidified air with 5% CO₂. Two culture wells were employed for each experiment, including one for the experimental group and the other one for the control group.

Short hairpin (sh)RNA (pGPU6) targeting A20 (sh-A20#1, 5'-GCAACTGGAGTCTCTCAAATC-3' and sh-A20#2, 5'-TTTGAAAGTGGGTGGAATTTA-3') and its corresponding negative control (sh-NC, 5'-GCAACAAGATGAAGAGCACCAA-3') were designed and provided by Guangzhou RiboBio Co., Ltd. MDA-MB-231 cells were transfected with sh-NC or sh-A20#1/2 (1 µg) using Lipofectamine^® 2000 for 48 h at 37˚C in 5% CO₂.

THP-1 cells were seeded into 6-well plates (5x10⁵ cells/well) and incubated with 200 ng/ml phorbol 12-myristate 13-acetate (PMA; MedChemExpress) for 24 h. Next, THP-1 cells were respectively cultured in PBS, or medium from MDA-MB-231 cells transfected with sh-NC, or medium from MDA-MB-231 cells transfected with sh-A20 for 24 h. Finally, THP-1 cells were treated with the NLRP3 inhibitor MCC950 (1 µM; MedChemExpress) for 1 h at 37˚C.

Total RNA from MDA-MB-231 cells subjected to different treatments was extracted with TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was then reverse transcribed into cDNA with RevertAid First-Strand cDNA Synthesis Kit (Takara Bio, Inc.). Sequentially, ~150 ng cDNA was used for qPCR with SYBR^® Premix Ex Taq II RT-PCR Kit (Takara Bio, Inc.) in a RT-qPCR system. The thermocycling conditions were as follows: i) 95˚C for 3 min; and ii) 40 cycles of 95˚C for 15 sec, 60˚C for 35 sec and 72˚C for 25 sec. The relative quantification of A20, CD86, CD80, inducible nitric oxide synthase (iNOS), CD206, CD163, CD11b, arginase 1 (ARG1) and IL10 was analyzed by the 2^-ΔΔCq method (27). GAPDH was used as an endogenous control. The primer sequences used in the present study were as follows: A20, forward 5'-TCAAAATGGCTTCCACAGACAC-3' and reverse, 5'-GTCCTTCAGGGTCACCAAGG-3'; CD86, forward 5'-AGCCCACAGGAATGATTCGC-3' and reverse, 5'-TCTGCATAACACCATCATACTCGA-3'; CD80, forward 5'-CGCCTCTCTGAAGATTACCCAAA-3' and reverse, 5'-TAAGACCAGGGCACTTCCCA-3'; iNOS, forward 5'-GATCAAAAACTGGGGCAGCG-3' and reverse, 5'-CTCATCTGGAGGGGTAGGCT-3'; CD206, forward 5'-CATCAGGGTGCAAGGAAGGT-3' and reverse, 5'-TCCATCCGTCCAAAGGAACG-3'; CD163, forward 5'-GAAGACAGAGACAGCGGCTT-3' and reverse, 5'-GGTATCTTAAAGGCTCACTGGGT-3'; CD11b, forward 5'-GCTTTGGTGGCTTCCTTGTG-3' and reverse, 5'-TAGTCGCACTGGTAGAGGCT-3'; ARG1, forward 5'-ACTTAAAGAACAAGAGTGTGATGTG-3' and reverse, 5'-CATGGCCAGAGATGCTTCCA-3'; IL-10, forward 5'-TTGCAAAACCAAACCACAAGACA-3' and reverse, 5'-TCTCGAAGCATGTTAGGCAGG-3'; and GAPDH, forward 5'-AATGGGCAGCCGTTAGGAAA-3' and reverse, 5'-GCGCCCAATACGACCAAATC-3'.

MDA-MB-231 cells from different groups were suspended in PBS and lysed with RIPA buffer (Cell Signaling Technology, Inc.), followed by centrifugation at 10,000 x g for 10 min at 4˚C to obtain total protein. The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). The protein samples (30 µg per lane) were separated by SDS-PAGE (10% gels; Bio-Rad Laboratories, Inc.) and then transferred to PVDF membranes (Bio-Rad Laboratories, Inc.) The membranes were blocked with 5% non-fat milk for 2 h at room temperature, and incubated with primary antibodies against A20 (cat. no. ab92324; dilution, 1:1,000; Abcam), VEGFA (cat. no. ab214424; dilution, 1:1,000; Abcam), NLRP3 (cat. no. ab263899; dilution, 1:1,000; Abcam), cleaved caspase-1 (cat. no. #4199; dilution, 1:1,000; Cell Signaling Technology, Inc.), IL-1β (cat. no. ab254360; dilution, 1:1,000; Abcam), caspase-1 (cat. no. #2225; dilution, 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat. no. ab8227; dilution, 1:1,000; Abcam) overnight at 4˚C. The following day, the membranes were washed with 0.05% Tween-20/1X TBST (cat. no. T1085; Beijing Solarbio Science & Technology Co., Ltd.) three times for 10 min each at room temperature and then incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (cat. no. ab205718; dilution, 1:2,000; Abcam) for 1 h at room temperature. The protein bands were visualized using enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.) and quantified by ImageJ 1.8.0 software (National Institutes of Health).

MDA-MB-231 cells from different treatment groups were seeded into 96-well plates (2x10³ cells/well), and incubated at 37˚C with 5% CO₂ for 24, 48 and 72 h. CCK-8 reagent (10 µl; MedChemExpress) was added per well after the indicated time, followed by incubation at 37˚C for another 1 h. The absorbance values were recorded in a microplate reader (Bio-Rad Laboratories, Inc.) at 450 nm.

MDA-MB-231 cells from different treatment groups were seeded into 6-well plates (1,000 cells/well) and then cultured in DMEM at 37˚C for 14 days. Next, the colonies were fixed with 4% paraformaldehyde at room temperature for 15 min and stained with 0.1% crystal violet at room temperature for 15 min, followed by observation under an Olympus digital camera (Olympus Corporation).

MDA-MB-231 cells from different treatment groups were seeded in a 6-well plate (1x10⁵ cells) and cultured until 80% confluence. The cell layers were scratched with a 100-µl pipette tip, and the wells were then washed with PBS to remove cell debris. Next, the cells were incubated with serum-free DMEM for 24 h at 37˚C, and photographed under a light microscope (Olympus Corporation). The extent of wound healing was calculated as follows: Wound healing (%)=(wound area at 0 h-wound area at 24 h)/wound area at 0 h.

MDA-MB-231 cells (1x10⁵ cells) from different treatment groups were seeded in the upper chamber of a Transwell plate (8.0-µm pore membranes, 24 wells; Corning, Inc.) that had been coated with Matrigel for 30 min at 37˚C, while complete medium was added to the lower chamber. After incubation for 24 h at 37˚C, the cells inside the upper chamber were removed, and the cells outside the upper chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution for 5 min at room temperature. The invasive cells were observed and photographed under a light microscope (Olympus Corporation).

HUVECs were seeded on a 24-well plate (7x10⁵ cells/well) coated with Matrigel (BD Biosciences) and incubated in endothelial cell growth medium from Control, sh-NC and sh-A20 groups for 48 h at 37˚C. Tube formation was visualized under a light microscope (Olympus Corporation), and the number of tubes was calculated using the Image-Pro Plus software (v6.0; Media Cybernetics, Inc.).

To identify the polarization states of macrophages (M1 co-expressing F4/80 + iNOS and M2 co-expressing F4/80 + ARG-1), double staining immunofluorescence was performed. After blocking with 3% BSA (Thermo Fisher Scientific, Inc.) at room temperature for 30 min, THP-1 cells were incubated with anti-F4/80 (cat. no. sc-377009; dilution, 1:100; Santa Cruz Biotechnology, Inc.), anti-iNOS (cat. no. sc-7271; dilution, 1:100; Santa Cruz Biotechnology, Inc.) and anti-ARG-1 (cat. no. #93668; dilution, 1:50; Cell Signaling Technology, Inc.) antibodies. Next, THP-1 cells were washed with PBS and incubated with goat anti-mouse IgG H&L (Alexa Fluor^® 488) secondary antibody (cat. no. ab150113; dilution, 1:200; Abcam) at room temperature for 1 h in the dark. Next, the cells were washed with PBS and incubated with DAPI at room temperature for 10 min in the dark. Finally, THP-1 cells were observed and photographed under a fluorescence microscope (Olympus Corporation).

Data are presented as the mean ± SD from ≥3 experiments and analyzed by GraphPad Prism 1.8.0 program (GraphPad Software; Dotmatics). One-way ANOVA followed by Tukey's post hoc test was applied to analyze the differences between groups. P<0.05 was considered to indicate a statistically significant difference.

The expression of A20 in breast cancer cells was increased compared with that in MCF-10A cells, and A20 expression was the highest in MDA-MB-231 cells; therefore, the MDA-MB-231 cell line was selected for subsequent experiments (Fig. 1A and B).

When MDA-MB-231 cells were transfected with sh-A20#1 and sh-A20#2, the expression of A20 was decreased, and was the lowest in sh-A20#2 group, which was therefore selected for the following experiments (Fig. 1C and D). MDA-MB-231 cells transfected with sh-A20 showed decreased viability (Fig. 1E). The proliferation of MDA-MB-231 cells was suppressed by interference with A20 (Fig. 1F and G).

The invasion and migration of MDA-MB-231 cells were inhibited by interference with A20 (Fig. 2A-D). The tube formation of HUVECs was suppressed in the culture medium from sh-A20-transfected MDA-MB-231 cells (Fig. 2E). The expression of VEGFA in HUVECs cultured in culture medium from sh-A20-transfected MDA-MB-231 cells was decreased (Fig. 2F).

After treating THP-1 cells with PMA, THP-1 cells cultured in PBS showed a high percentage of M1-like polarization of macrophages. When PMA-treated THP-1 cells were cultured in culture medium from sh-NC-transfected MDA-MB-231 cells, M1-like polarization of macrophage marker iNOS expression was decreased, while M2-like polarization of macrophage marker ARG1 expression was increased. When PMA-treated THP-1 cells were cultured in culture medium from sh-A20-transfected MDA-MB-231 cells, NOS expression was increased, while ARG1 expression was declined (Fig. 3A). Correspondingly, the levels of CD86, CD80 and iNOS, which are related to M1-like polarization of macrophages, were lower in the in the MDA-MB-231 sh-NC group compared with the control group, while the levels of CD206, CD163, CD11b, ARG1 and IL10, which are related to M2-like polarization of macrophages, were increased in the MDA-MB-231 sh-NC group. The levels of CD86, CD80 and iNOS (related to M1-like polarization of macrophages) were upregulated, while the levels of CD206, CD163, CD11b, ARG1 and IL10 (related to M2-like polarization of macrophages) were downregulated in the MDA-MB-231 sh-A20 group (Fig. 3B and C).

The expression of NLRP3, cleaved caspase-1 and IL-1β in THP-1 cells cultured in culture medium from sh-NC-transfected MDA-MB-231 cells was decreased. However, the culture medium from sh-A20-transfected MDA-MB-231 cells increased the levels of NLRP3, cleaved caspase-1 and IL-1β in THP-1 cells (Fig. 4).

MCC950 suppressed the levels of CD86, CD80 and iNOS (related to M1-like polarization of macrophages), and enhanced the levels of CD206, CD163, CD11b and IL10 (related to M2-like polarization of macrophages) (Fig. 5A and B). The culture medium from sh-A20-transfected MDA-MB-231 cells suppressed the proliferation of THP-1 cells, which was reversed by MCC950 (Fig. 5C). The invasion ability of THP-1 cells cultured in culture medium from sh-A20-transfected MDA-MB-231 cells was inhibited, and MCC950 restored the invasion ability of THP-1 cells cultured in culture medium from sh-A20-transfected MDA-MB-231 cells (Fig. 5D).