br volume Dmax Dmin Three weeks after
volume = 0.5 × (Dmax × Dmin2). Three weeks after injection, the mice were sacrificed, and the primary tumours, livers and lungs were removed for further evaluation.
2.13. Statistical analysis
All experiments were performed in triplicate. All data were statis-tically analyzed using SPSS version 17.0, and graphs were made using GraphPad Prism version 5.0 software. One-way ANOVA and Newman-Keuls post hoc tests were used to compare variances between groups. P value < .05 was considered to be a significant diﬀerence.
Fig. 3. ASCs/SCF inhibit miR20b transcription in BC cells through the p-c-Kit/p38 MAPK/E2F1 pathway. (A) 4T1 cells were stimulated with SCF (50 ng/ml or 100 ng/ml) or (B) 4T1 cells were transfected with an siRNA specific for MEK (si-MEK) or p38 (si-p38) or a control siRNA and then the indicated proteins were analyzed by western blot. (C) Basic information and location of the predicted E2F1 2-Guanidinoethylmercaptosuccinic Acid on the miR106a-363 cluster promoter. (D) Relative luciferase activity of pGL3-R1 and pGL3-R2 after pcDNA3.1-E2F1 or pcDNA3.1-EGFP transfection. (E-F) 4T1 cells transfected with p38-specific siRNA were cocultured with ASCs and then E2F1 protein binding to the miR106a-363 cluster promoter was analyzed using a ChIP assay. *P < .05, **P < .01, ***P < .001, NS not significant.
3.1. SCF released by c-kit+ ASCs contributes to the induction of BC cell migration and invasion through HIF-1α and VEGFA activation
To confirm the eﬀects of ASCs on the migration and invasion of BC cells, we cultured 4T1 breast cancer cells with the supernatant of ASC-
conditioned medium. The results showed that the migration and inva-sion abilities of 4T1 cells were significantly enhanced by the ASCs (Fig. 1A–B). ASCs produce several cytokines and chemokines that aﬀect cancer cells . To explore the molecular mechanisms of the ASCs enhanced the migration and invasion of BC cells, we analyzed the levels of SCF, IL-6, sCD31, monocyte chemotactic protein-1 (MCP-1), hypoxia inducible factor 1-α (HIF-1α), macrophage inflammatory protein-1α
(MIP-1α), stromal cell-derived factor-1 (SDF-1), vascular endothelial growth factor A (VEGFA) and tumor necrosis factor α (TNFα) in direct ASC/4T1 coculture (see online supplementary Table S1–3).
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Quantification analysis revealed that the levels of SCF, HIF-1α and VEGFA were significantly higher in the ASC/4T1 coculture group than in the single culture groups (Fig. 1C–E). Pretreatment of ASCs with an
Fig. 4. ASCs activates HIF-1α and VEGFA by silencing miR20b in BC cells. (A-B) Luciferase reporter plasmids containing the wild-type or mutant miR20b binding sequence in the 3′-UTRs of HIF-1α and VEGFA mRNA. The luciferase plasmids and a control vector or a miR20b agomir were cotransfected into 4T1 cells and then the relative luciferase activity was analyzed in the cells. (C) The untransfected 4T1 cells or 4T1 cells transfected with a miR20b agomir were cocultured with ASCs and chromatin immunoprecipitation assays were performed using anti-HIF-1α and RNAPII; the amplified products of the VEGFA promoter were analyzed by agarose gel electrophoresis. IgG was used as a negative control. (D) The location of HIF-1α (green fluorescence) in 4T1 cells was assessed by confocal microscopy. (E-F) 4T1 cells were transfected with miR20b agomir, miR20b agomir plus HIF-1α, HIF-1α vector or control constructs for 24 h and then cocultured with ASCs for 24 h. The migration (E) and invasion (F) of these cells were assessed using a transwell assay and western blot. *P < .05, **P < .01, ***P < .001, NS, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
anti-SCF antibody (isotypic IgG was used as a control at 10 μg/ml) re-sulted in the inhibition of ASC-induced migration and invasion of 4T1 cell, and mouse recombinant SCF (100 ng/ml) induced 4T1 cell mi-gration and invasion (Fig. 1F–G). Moreover, treatment with an anti-SCF antibody resulted in the inhibition of ASC-induced HIF-α and VEGFA expression in 4T1 cells (Fig. 1H, Supplementary Fig. S1A–B). These results indicate that SCF secreted from ASCs predominantly activates HIF-α and VEGFA and mediates the ASC-induced migration and inva-sion of the BC cells.
3.2. ASC-induced BC cell migration and invasion are mediated by SCF-dependent -induction of miR-20b downregulation in BC cells
Since miR20b is negatively correlated with the advanced invasive stages of breast cancer as an antisense-miR, we examined the con-tribution of miR20b to the ASC-induced migration and invasion of 4T1 cells by coculture of 4T1 cells with ASCs or by treatment of 4T1 cells with SCF, and analyzed the expression levels of primary/precursor and mature miR20b in 4T1 cells. We observed that the expression levels of miR20b in 4T1 are downregulated after coculture and treatment with SCF (Fig. 2A–B). Furthermore, the overexpression of miR20b inhibited ASC-induced migration and invasion of 4T1 cells (Fig. 2C–D). To fur-ther investigate the eﬀects of miR20b on HIF-1α and VEGFA protein expression levels in BC cells, a miR20b agomir or a miR20b inhibitor were used to upregulate or knockdown the expression of miR20b, re-spectively. We found that transfecting 4T1 cells with a miR20b agomir inhibited ASC-induced HIF-1α and VEGFA expression; however, in-tracellular HIF-1α and VEGFA protein levels increased after miR20b silencing (Fig. 2E, Supplementary Fig. S1C–D). Pretreatment of 4T1 cells with an anti-SCF antibody resulted in the upregulation of ASC-induced miR20b (Fig. 2F). These results suggest that ASC-induced breast cancer cell migration and invasion are mediated by SCF-depen-dent induction of miR20b downregulation and subsequent regulation of HIF-1α and VEGFA expression in BC cells.