The human ovarian cancer cell lines SKOV3, OVCAR3, A2780 and Caov3 were obtained from the America Type Culture Collection (ATCC; Manassas, VA, USA), and HO-8910 cell lines, Hosepic cell lines and THP-1 cell lines were obtained from the China Center for Type Culture collection (CCTCC). The cell lines were cultured in either Dulbecco's modified Eagle's medium (DMEM, Hyclone, Cat.No.SH30022.01B) or RPMI-1640 medium (Hyclone, Cat.No.SH30809.01B) supplemented with 10% fetal bovine serum (FBS, Hyclone,Cat.No.SH30256.01B) and antibiotics (penicillin 100 U/mL, streptomycin 0.1 mg/mL and amphotericin B 0.25 lg/mL) and maintained in a 37̊C incubator containing 5% CO2. THP-1 cells was treated with 10 ng/ml phorbol 12-myristate 13-aceteat (PMA; SigmaAldrich, Germany) for 72 h to induce differentiation of M0 macrophage. M0 macrophages were treated with 100 ng/ml LPS (#L2880, sigma) plus 10 ng/ml IFN-γ (#BEK-2026, 4A Biotech, Beijin) for 48 h to induce M1 phenotype differentiation or with 10 ng/ml IL-4 (#214 − 14, Peprotech, Germany) for 48 h to induce M2 phenotype differentiation. Moreover, M0 macrophages were also treated with the supernatant of each ovarian cancer cell line for 48 h. Cells were collected for flow cytometry analysis, RT-qPCR or Western blot assays.
Real-time quantitative PCR(RT-qPCR).
Total RNA was extracted from cells using TRIzol reagents (Pufei Biotechnology, Shanghai, China). Reverse transcription was performed using M-MLV reverse transcriptase (Promega, Madison, USA).
The primer sequences were designed as follows:
Quantitative PCR was performed using SYBR-Green RealTime PCR Master Mix (Toyobo,Osaka,Japan) according to the manufacturer's protocol. Data were analyzed by Sequence Detection Software for the threshold cycle(Ct), and the comparative Ct (ΔΔCt) was used to calculate the difference between samples by relative qualification.
Total cell lysates were harvested in NP-40 lysis buffer (150 mM NaCl, 1% Nonidet P-40, 50 mM Tris, pH 8.0, protease inhibitor cocktail) and protein concentrations were determined by BCA protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of proteins from each lysate were submitted to SDS-PAGE for protein separation and then transferred to PVDF membranes. Membranes were blocked with buffer containing 5% skim milk and 0.1% Tween-20 in PBS for 1 h at room temperature with gentle shaking. Primary antibodies (FPR2,RhoA and GAPDH) were incubated overnight at 4˚C with gentle shaking, followed by secondary antibody incubation at room temperature for 1 h with gentle shaking. The following antibodies were used: FPR2 (#sc-66898; Santa Cruz Biotechnology, Santa Cruz, CA, USA); NF-κB (#ab54835; Abcam, Cambridge, MA, USA); GAPDH (#KC-5G5; AKsomics,ShangHai); Goat Anti-Rabbit IgG (H + L), ads-HRP (#4050-05; southern biotech, Birmingham, AL, USA ).
Vector construction and plasmid transfection.
PCR product and pcDNA3.1 vector (invitrogen) were both treated by double digestion of XhoI and HindIII. Target fragments were separated and purified, and the recombinant plasmid was constructed. T4-DNA ligase was used to combine the vector and the target gene. Caov-3 cell lines and OVCAR-3 cell lines were transefected with pcDNA3.1-shFPR2 vector and pcDNA3.1-FPR2 + vector respectively; as well as both of the corresponding control cell lines were constructed. The shRNA sequences for FPR2 knock down were as follows: shRNA-, ccggGGCCAAGACTTCCGAGAGAGActcgagTCTCTCTCGGAAGTCTTGGCCtttttg; The FPR2 over expressed RNA sequence were: TCACCTCCTGCAGAGACTGAGTTACAGGCAATGTGA.
Concentration of cytokines including TGF-β, IL-4, and IL-10, was determined using ELISA kits from Solarbio (Cat. No. #SEKH0316, #SEKH0011, and #SEKH0018, respectively). Cell culture supernatants were collected and centrifuged at 4 °C at 1000 × g for 10 min before analyzed according to the manufacturer's protocol.
For fow cytometric analysis, cells were stimulated as described above. 1 × 106/cells per sample were collected and stained with antibody in 100 µl PBS + 1% BSA for 30 min at 4 °C in the dark, followed by wash with PBS. Fuorescence was detected on a BD FACSCan (Becton, Dickinson and Company, MD, USA). All antibodies, except Dectin-PE (R&D Systems, Germany), were obtained from BD Bioscience (New Jersey,America).
Cells (3 × 104 /well) were seeded on 96-well plates and grown to 90% confluence, after which a scratch was made in the monolayer using a 10-µl pipette tip. Then, the cells were incubated at 37℃ in 5% CO2 for another 4 h according to the result of the pre-experiment, and the images were obtained at the time points of 0, 4 and 8 h. Each experiment was performed three times.
The assay was performed using a pre-coated cell invasion kit (pore size, 8.0 µm; Corning Inc., Corning, NY, USA) and Matrigel (BD Biosciences, Bedford, MA, USA) was inserted in the upper chambers. Approximately 1 × 105 cells in 100 µl serum-free medium were placed into the upper chambers, the cells were cultured in 5% CO2 at 37℃ for 16 h (according to the pre-experiment). The lower chambers contained 30% FBS, thus, the cells migrated to the lower chambers. The cells remaining in the upper chambers were removed with a cotton swab and the cells that migrated through the membrane to the lower surface were stained with Giemsa's staining for 3–5 min at room temperature. The number of cells that migrated through the lower membrane of the inserts was counted under a light microscope. Each experiment was performed three times.
Statistical analysis was performed using IBM SPSS Statistics 23.0 (IBM SPSS, Armonk, NY, USA). Statistics of continuous data were performed using AVOVA or Kruskal-Wallis test; correlation analyses were performed using Pearson correlation analysis. At least three independent experiments for each group were conducted, and differences between groups were assessed by variance analysis and Student's t-test. P-value < 0.05 was considered to indicate a statistically significant result.