br RNA oligonucleotides and plasmid transfection br
2.8. RNA oligonucleotides and plasmid transfection
Breast cancer Malonyl Coenzyme A were respectively transfected with miR20b agomir, miR-20b inhibitor, MEK siRNA, P38 siRNA or nonspecific siRNA (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol. The se-quences of the siRNA oligonucleotides or shRNA were provided in on-line supplementary table S5. At 48 h after transfection, RNA and protein were isolated, and the relation between MAPK and E2F1 or E2F1 and miR20b promoter was tested.
2.9. Dual-luciferase reporter assay
4T1 breast cancer cells were co-transfected with the miR106a-363 (PGL-R1, PGL-R2) promoter-luciferase reporter plasmid (GenePharma, Shanghai, China) and E2F1 overexpression vector or control vector, and the pRL Renilla reporter (Promega, USA) as a control for the promoter assays of pri-miRNAs.
To validate HIF-1α and VEGFA as a direct target of miR20b, 4T1 cells were co-transfected with wild-type or mutant HIF-1α/VEGFA 3′UTR dual-luciferase reporter with miR20b agomirs or NC vector using Lipofectamine 2000 reagent in 96-well plates. After 24 h, cells were harvested, and luciferase activity was quantified using a Dual-Luciferase Assay Kit (Promega Corp., WI).
2.10. Immunofluorescence staining
Adherent cells plated on EZ Slides (Millipore, USA) were labelled with rat monoclonal anti-HIF-1α antibody (Abcam), followed by donkey anti-rat secondary antibody conjugated with Alexa Fluor 488 (Thermo Scientific, USA). After incubation with 4′6-diamidino-2-phe-nylindole (Thermo Scientific, USA) for 1 min, the cells were observed under a confocal microscopy system (Yokogawa, Tokyo, Japan).
2.11. Chromatin immunoprecipitation assay (ChIP)
ChIP was conducted using a ChIP assay kit (Active Motif, Rixensart,
Fig. 1. ASC-induced migration and invasion of BC cells is mediated by SCF-dependent activation of HIF-1α and VEGFA. (A) Wound healing (B) and transwell assays were used to detect the migration and invasion of 4T1 cells treated with ASC culture supernatant. (C–E) The release of cytokines and chemokines including SCF, IL-6, sCD31, MCP-1, HIF-1α, MIP-1α, SDF-1, VEGFA and TNFα in 4T1 cells cocultured with ASCs was analyzed using a Milliplex MAP kit. (F) The migration (G) and invasion of 4T1 cells (H) and activation of HIF-1α and VEGFA induced by ASCs were assessed. The cells were pretreated with IgG or anti-SCF neutralizing antibody for 1 h. The cells stimulated with SCF for 2days were used as a positive control. *P < .05, **P < .01, ***P < .001, NS, not significant.
Belgium) following the manufacturer's instructions. Briefly, the cells were fixed with 1% formaldehyde for 10 min, after which chromatin was sonicated and immunoprecipitated with anti-HIF-1α antibodies, anti-E2F1 antibodies or non-immune rabbit IgG (Abcam, USA), and 10% chromatin was used as an input control. The immunoprecipitated
DNA was assessed for the presence of the VEGFA promoter domain by PCR using the following primers: VEGFA forward 5′-CAGGAACAAGG GCCTCTGTCT-3′, reverse 5′-TGTCCCTCTGACAATGTGCCATC-3′; miR106a-363(1698 bp) forward: 5′- GAGAATGGAAAGATAAACA CGG-3′, reverse: 5′-TTTTGGCTGGTCGGTGC-3′; miR106a-363(2847 bp)
Fig. 2. ASC/SCF-induced BC cell migration and invasion are mediated by the inhibition of miR20b. (A) qPCR analysis of the expression levels of the primary/ precursor and mature forms of miR20b in 4T1 cells cocultured with ASCs (B) or in 4T1 cells pretreated with SCF (50 ng/ml or 100 ng/ml) for 48 h. (C–E) 4T1 cells were transfected with a miR20b agomir or the corresponding control for 24 h and then cocultured with ASCs for 24 h. The cells pretreated with a miR-20b inhibitor were used as a positive control. The migration (C) and invasion (D) of the cells and the activities of HIF-1α and VEGFA (E) were assessed using a transwell assay and western blot, respectively. (F) The 4T1 cells pretreated with an anti-SCF antibody were cocultured with ASCs and then the expression levels of miR20b were determined by qPCR. *P < .05, **P < .01, ***P < .001, NS, not significant.
forward: 5′-CCGGTCCTGCCATGTTT-3′, reverse: 5′-ACGCTTCCACTGC TCCTG-3′. The amplified product was analyzed by agarose gel elec-trophoresis.
Subcutaneous orthotopic injection in 4-week-old female nude mice (BALB/c) was performed under intraperitoneal injection anaesthesia (1.2% Avertin, 0.1 ml/10 g). 4T1 cells, and miR20bup 4T1 cells were resuspended in 200 μl of PBS/Matrigel and injected into the mammary fourth fat pads of female nude mice with ASCs. In each group (n = 5), the tumor size was measured twice per week, and the tumor volume was calculated according to the following formula: tumor