The human monocytic cell line THP-1 and human breast cancer cell line MDA-MB-231 were purchased from American Type Culture Collection (ATCC) and cultured in the Dulbecco's modified Eagles' medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. To induce M2-type macrophages, THP-1 were pretreated with phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 18 h, and then cultured by IL-4 (25 ng/mL) for 3 d. For the following experiments, both M2-type macrophages and THP-1 cells were planted in the 96-well plates with 2 × 105 cells/mL, 200 µL/well. After 24 h, the supernatant (100 μL) was collected to culture MDA-MB-231 cells for 24 h.
Total RNA was isolated from cells using Trizol reagent (Invitrogen) and quantified by 10% agarose gel electrophoresis. The extracted RNA was used to reverse-transcribe into cDNA. For real-time PCR, cDNA was used as the template, and SYBR Green Real-time PCR Master Mix (Toyobo, Japan) was used for the experiments based on an ABI 7500 real-time PCR system (Applied Biosystems). The mRNA expression was assessed using 2−ΔΔCt method. GAPDH served as internal control.
Cells were treated by RIPA buffer and centrifuged at 15,000 g under 4°C for 15 min. The collected protein concentration was determined by BCA protein assay kit (Invirtogen). After separated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred onto PVDF and incubated with the primary antibodies of Arg-1, CD86, CD206, iNOS, IRF5, IRF6, IRF7, IRF9 and GAPDH (Abcam) at 4°C for 24 h. After washed by PBS for 3 times, the PVDF was incubated with the secondary antibody (Beyotime, Shanghai, China) at room temperature for 1 h. The protein bands were visualized by ECL methods. GAPDH severed as internal control.
Cell proliferation was determined using Cell Counting Kit-8 (CCK-8, Sangon Biotech, Shanghai, China). Briefly, cells were planted at a 96-well plate with the concentration of 103 cells per well. After 24 h, the cells were incubated with CCK-8 (10 μL) solution at 37 °C for 3 h. A microplate reader was employed to detect the absorbance at 490 nm (Bio-Rad, CA, USA).
For determination cell migration, scratch wound healing assays were performed. Briefly, cells were seeded in a 6-well plate with the concentration at 105 cells per well. After 25 h, cells were spread across the plate, and 200 μL micropipette tip was used to scratch through the cells. After washing by PBS, cells were cultured at 37°C with 5% CO2. After 24 h, cell migration was photographed with an inverted microscope (40 × magnifications, Olympus, Japan).
The inhibitor and the mimic of miR-1587 and their negative control were purchased from Sangon Biotech (Shanghai, China). The pcDNA-IRF7, si-IRF7 and their negative control were purchased from RiboBio (Guangzhou, China). After reached to 70% influence, cells were transfected with the Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instruction. After 24 h, the transfection efficiency was determined.
Dual-luciferase reporter assay
The fragment of IRF7 3’UTR containing the predicted binding sites of miR-1587 wild-type (WT) or mutant (MUT) was amplified and cloned into pmirGLO luciferase vector (Promega, USA). Next, the miR-1587 mimic or its negative control was co-transfected with the reporter plasmid into HEK293 cells. After 48 h, the luciferase activity was determined using a dual-luciferase reporter assay system (Promega, USA) following the manufacturer’s instruction.
A total of 18 female BALB/c nude mice (6-weeks old) were purchased from Vital River (Beijing, China). Mice were raised under standard condition at 25°C, and 12-12 h dark-light cycles. All mice were randomly divided into 3 groups (n=6 in each group), including control, M2 and M2+miR-1587(-). In control group, mice were subcutaneously injected MDA-MB-231 cells (5×106 cells/200 μl PBS/mouse). In the M2 group, MDA-MB-231 cells were co-cultured with the supernatant medium of M2-type macrophages for 24 h, and then subcutaneously injected into mice. In the M2+miR-1587 (-) group, MDA-MB-231 cells were transfected with anti-miR-1587 and then co-cultured with the supernatant medium of M2-type macrophages for 24 h, after that, the cells were subcutaneously injected into mice. After 7 d, the supernatant medium of M2-type macrophage was injected into tumor (100 μL/mouse), and the injection was performed every three days. Tumor volumes were measured once per week for 4 weeks. Four weeks later, all mice were euthanasia, and tumor tissues were collected for determination of miR-1587 and IRF7 expression.
The study was approved by the Ethics Committee of Huai'an Second People's Hospital and the Affiliated Huai'an Hospital of Xuzhou Medical University, and all experiments were handled in accordance with the animal care and use guidelines.
All experiments were repeated for three independent times. Statistical analysis was performed based on the SPSS 20.0 and GraphPad prism 8.0 software. Data were presented as means ±SD. Statistical difference between two groups was analyzed using Student t-test, while one-way analysis (ANOVA) was conducted to determine difference among three or more groups. P<0.05 was considered as statistically significant difference.