br with miR located within the
with miR-17 located within the miR-17-92 cluster and the miR-106b-25 cluster . It has been reported that an autoregulatory feedback loop between E2F1 and miR20b-5p is involved in cell proliferation and diﬀerentiation . However, only a few studies have investigated whether miR20b mediates the epithelial to mesenchymal transition (EMT) and metastasis in breast cancer, especially in the context of regulation of breast cancer cell metastasis by ASCs.
Herein, we found that ASCs inhibited the biogenesis of miR20b in breast cancer gamma-Glu-Cys through the paracrine release of SCF with the sub-sequent induction of breast cancer cell migration and invasion. Mechanistically, we demonstrated that the p-c-Kit/MAPK-p38/E2F1 cascade is involved in ASC/SCF-induced miR20b downregulation in BC cells. Furthermore, ASC induction of miR20b downregulation activated HIF-1α/VEGFA and induced EMT and metastasis of BC cells in vitro and in vivo.
2. Materials and methods
2.1. Cell lines and reagents
The breast cancer cell lines 4T1 were obtained from the American Type Culture Collection (Manassas, USA). All of the medium were purchased from HyClone and supplemented with 10% FBS (Gibco, USA). ASCs were isolated and identified as previously described . SCF was purchased from R&D Systems and utilized at a final con-centration of 100 ng/ml.
Four-week-old female nude mice (BALB/c) were obtained from the SLAC Laboratory Animal Center (Shanghai, China) and were housed in a specific-pathogen-free room. All the experimental protocols and an-imal handling procedures were approved by the Animal Committee of Harbin Medical University. All experimental procedures and post-operative animal care were conducted in accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals.
2.3. Wound healing assay
Breast cancer cells were plated into a 6-well plate. A scraped line was created using a 200 μl pipette tip when the cells had achieved 100% confluence. After incubation for 24 h, the wound closure was imaged with a microscope and the rate of closure was assessed.
After coating the wells of a 24-well transwell culture plate (Corning, NY, USA) with 100 μl Matrigel (1 mg/ml) (BD, NY, USA), untransfected or transfected 4T1 cells were resuspended in FBS-free medium and plated in the upper chamber; the bottom chamber was filled with FBS-free medium with or without ASCs. The plates were incubated for 24 h at 37 °C and 5% CO2. After removing the cells from the upper side of the membrane, the membrane was fixed with 4% formaldehyde solution and stained with 1% crystal violet solution. Then, the cells on the bottom side of the membrane were counted in five randomly chosen fields of view using a microscope at 200 × magnification.
2.5. Quantification of cell supernatant cytokines
Supernatant medium was collected after culturing for 1, 3, and 5 days by centrifugation for 10 min at 1000 rpm. The supernatant was immediately subjected to cytokine and chemokine detection with a commercially available Milliplex MAP kit (Cat.MAGPMAG-24 K, MMMP3MAG-79 K, Millipore Corporation, Billerica, MA, USA), ac-cording to the manufacturer's protocols. Cellular Signalling 62 (2019) 109350
2.6. RNA extraction and quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted using RNeasy Mini Kits (QIAGEN, Germany), according to the manufacturer's protocol. Total RNA (1 μg) was reverse-transcribed into cDNA using a QuantiTect Reverse Transcription Kit (QIAGEN). The primer sequences were provided in online supplementary table S4. qPCR was performed as described pre-viously . Amplification parameters were as follows: one cycle of 50 °C for 2 min and 95 °C for 3 min, followed by 35 cycles of 95 °C for 10 s and 60 °C for 30 s. Relative expression levels were determined using the comparative threshold cycle method (2- Ct).
All primary antibodies were obtained from Abcam (Abcam, USA). Proteins from cell and tissue lysates were analyzed using Western blotting as described previously , in which the nuclear protein ex-traction was performed according the protocol of CelLytic™ NuCLEAR™ Extraction Kit (Sigma, USA). In brief, samples were incubated overnight at 4 °C with primary antibodies against HIF-1α (1:1000), VEGFA (1:2000), c-Kit (1:500), p-c-Kit (1:1000), p-ERK1/2 (pT202/pY204, 1:1000), ERK1/2 (1:1000), P38(1:1000), E2F1 (1:2000), and then in-cubated with horseradish peroxidase (HRP)-conjugated secondary an-tibodies (1:3000) for 2 h at room temperature; protein-antibody com-plexes were visualized with an Enhanced Chemiluminescence Western Blotting Detection Kit (Beyotime Biotechnology, Shanghai, China) and an analysis system (Bio-Rad, USA).