Overexpression of the miR-200b/200a/429 cluster or the miR-141/200c cluster enhanced the migratory and invasive abilities of MDA-MB-231 cells
We first investigated alterations in the characteristics of two different breast cancer cell lines, MCF-7 and MDA-MB-231 cells, caused by the stable transduction of constructs encoding miR-200 family members and GFP using lentiviruses. The GFP-positive cells comprised more than 95 % of the miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MCF-7 and MDA-MB-231 cells (Additional file 1: Figure S1 and Additional file 2: Figure S2). Strong GFP expression in the miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MCF-7 and MDA-MB-231 cells was evaluated using microscopy (Fig. 1a and Additional file 1: Figure S1A). RT-PCR was used to evaluate expression levels of EMT markers, and the results showed that the overexpression of the miR-200b/200a/429 cluster or the miR-141/200c cluster resulted in the induction of E-cadherin expression and a decrease in ZEB-1 expression in MDA-MB-231 cells (Additional file 1: Figure S1B). Overexpression of the miR-200b/200a/429 or miR-141/200c cluster led to increased expression of Snail in MCF-7 and MDA-MB-231 cells.
To investigate the stable overexpression of miR-200a, miR-200b, miR-200c in miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells, real-time RT-PCR was conducted to quantify the levels of the mature microRNAs. As expected, the miR-200b/200a/429 cluster- and miR-141/200c cluster-transduced MDA-MB-231 cells showed increased mature miR-200a, miR-200b, and miR-200c levels at 5-100-fold higher levels than did the control MDA-MB-231 cells, which exhibited undetectable levels of all members of the miR-200 family (Fig. 1b).
To investigate whether overexpression of the miR-200b/200a/429 or miR-141/200c cluster affected cell growth, an MTT assay was performed. Figure 1c shows that the growth rate of the miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells was similar to that of the control cells on the 3rd day but was significantly decreased on the 5th day after cell seeding compared with that of the control cells (control vs. miR-200ab, p = 0.02 and control vs. miR-200c, p = 0.0002) (Fig. 1c). The overexpression of the miR-141/200c cluster was found to more strongly suppress the growth of MDA-MB-231 cells.
We assessed migratory and invasive abilities using a trans-well migration assay and a wound-healing assay. The results of crystal violet staining showed that the migratory ability of MCF-7 cells was suppressed by transduction of the miR-200 family, but there was no significant difference between that of the miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MCF-7 cells and control cells (Additional file 1: Figure S1C, E). The wound-healing assay, which demonstrated the lateral migratory ability of MCF-7 cells, yielded results similar to those of the trans-well migration assay (Additional file 1: Figure S1D, F).
With regard to the trans-well migration and invasion assays of the miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells, after 48 h of incubation without an FBS gradient, cell migration significantly increased up to 1.94 ± 0.22-fold and 2.49 ± 0.08-fold in miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells, respectively, compared with control cells (Fig. 1d control vs. miR-200ab, p = 0.04 and control vs. miR-200c, p = 0.002). To further investigate the migratory ability of miR-141/200c cluster-transduced MDA-MB-231 cells, the trans-well migration assay under the condition of a 10 % FBS gradient in the upper (0 % FBS) and the lower chamber (10 % FBS) was conducted for 24 h. A significant increase was observed in the migratory ability of the miR-141/200c-transduced MDA-MB-231 cells (1.53 ± 0.30-fold) relative to MDA-MB-231 cells (Additional file 3: Figure S3, p = 0.0009). As expected, the results of an invasion assay using Matrigel™ matrix-coated trans-well membranes showed a significant increase in the invasive ability of the miR-200b/200a/429 cluster-transduced cells (1.61 ± 0.31-fold) and miR-141/200c cluster-transduced cells (1.98 ± 0.40-fold) after 48 h of incubation compared with that of control cells (Fig. 1e, control vs. miR-200ab, p = 0.0005 and control vs. miR-200c, p = 0.0002). The elevated migration and invasion rates were higher in miR-141/200c cluster-transduced MDA-MB-231 cells than miR-200b/200a/429 cluster-transduced MDA-MB-231 cells. After 6 h of incubation, the transduction of miR-200b/200a/429 cluster or miR-141/200c cluster did not affect the lateral migratory ability of MCF-7 cells, but increased the lateral migration up to 2.78 ± 0.11-fold and 1.69 ± 0.11-fold in miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells, respectively (p = 0.02, Additional file 1: Figure S1F). In other TNBC cell lines, HCC-38 and Hs578T cells, we observed a significant increase in trans-well migration ability in both the miR-141/200c cluster-transduced HCC-38 cells (1.64 ± 0.11-fold, p < 0.001) and miR-141/200c cluster-transduced Hs578T cells (1.76 ± 0.44-fold, p = 0.0003) compared with the control cells (Additional file 4: Figure S4A, D). The mature miR-200c levels in miR-141/200c cluster-transduced HCC-38 and Hs578T cells were 252- and 205-fold higher than that in the control HCC-38 and Hs578T cells, which exhibited undetectable levels of miR-200c (p < 0.001, Additional file 5: Figure S5G, H).
We found that the overexpression of the miR-200b/200a/429 cluster or the miR-141/200c cluster resulted in the highest migratory capacity in MDA-MB-231 cells compared with the other TNBC cells, HCC-38 and Hs578T cells. Therefore, we focused on the regulatory mechanisms by which the miR-200b/200a/429 cluster or the miR-141/200c cluster promoted the migratory and invasive capacities of the MDA-MB-231 cell line, as a representative TNBC cell line.
Overexpression of the miR-200b/200a/429 cluster or the miR-141/200c cluster enhanced the phosphorylation of FAK and AKT and the expression of integrin in MDA-MB-231 cells
We investigated whether the overexpression of miR-200 family members modulated the focal adhesion kinase (FAK), PI3K/AKT, and MEK/ERK signaling pathways, which are involved in cell proliferation and migration. Representative western blotting results showed that the levels of FAK and AKT phosphorylation were greatly increased, but the level of ERK phosphorylation was not significantly changed in the miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells (Fig. 2a, left). The level of phosphorylated FAK was significantly increased up to 1.52 ± 0.26-fold and 3.12 ± 1.56-fold in the miR-200b/200a/429 cluster-transduced cells and miR-141/200c cluster-transduced cells, respectively (Fig. 2a, right, control vs. miR-200ab, p = 0.01 and control vs. miR-200c, p = 0.09). The level of phosphorylated AKT was also increased 2.21 ± 1.06-fold and 9.32 ± 11.07-fold in the miR-200b/200a/429 cluster-transduced cells and miR-141/200c cluster-transduced cells, respectively (Fig. 2a, right, control vs. miR-200ab, p = 0.19 and control vs. miR-200c, p = 0.32). We also observed that the miR-141/200c cluster significantly increased the phosphorylation levels of FAK (1.98 ± 0.37-fold, p = 0.04) and AKT (5.61 ± 1.73-fold, p = 0.04) in HCC-38 cells (Additional file 6: Figure S6C, D). Representative western blotting results showed that the expression levels of integrin-αV, which is associated with the FAK signaling pathway, were increased in both the miR-200b/200a/429 cluster- and miR-141/200c cluster-transduced cells (Fig. 2b, left). Integrin-αV expression was increased 1.57 ± 0.10-fold and 1.70 ± 0.24-fold in the miR-200b/200a/429 cluster-transduced cells and miR-141/200c cluster-transduced cells, respectively (Fig. 2b, right, control vs. miR-200ab, p = 0.01 and control vs. miR-200c, p = 0.04). Integrin-αV expression was also increased (13.61 ± 0.72-fold, p = 0.008) in miR-141/200c cluster-transduced HCC-38 cells (Additional file 6: Figure S6C, D). To investigate the cellular localization of phosphorylated FAK and integrin-αV, immunofluorescent staining was assessed. Phosphorylated FAK was largely localized at the focal adhesions of the plasma membranes of the miR-200b/200a/429 cluster- and miR-141/200c cluster-transduced cells (Fig. 2c). Integrin-αV accumulated and clustered at the periphery of the plasma membranes of the miR-200b/200a/429- and miR-141/200c-transduced cells compared with that in control cells (Fig. 2c). The levels of FAK phosphorylation and integrin-αV expression were higher in the miR-141/200c cluster-transduced HCC-38 cells than in the control cells (Additional file 6: Figure S6E).
To determine the signaling pathway involved in enhanced migratory and invasive ability of miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells, trans-well migration assays were performed in the presence of PF573228 or LY294002, which are chemical inhibitors of FAK and PI3K/AKT, respectively. The chemical inhibitors were added to both the upper and lower chambers, which would allow their effects to last throughout the entire experimental period. The enhanced migratory activity was completely inhibited in miR-200b/200a/429 cluster- and miR-141/200c cluster-transduced MDA-MB-231 cells treated with the FAK or PI3K/AKT inhibitor, reaching the basal migratory level of MDA-MB-231 cells (Fig. 2d). These observations demonstrated that the elevated migratory ability of MDA-MB-231 cells by stable overexpression of miR-200b/200a/429 cluster or miR-141/200c cluster was driven by the activation of the FAK and PI3K/AKT-mediated signaling pathways.
Overexpression of the miR-200b/200a/429 cluster or the miR-141/200c cluster increased the level of VEGF-A secretion in MDA-MB-231 cells
The secreted cytokines or growth factors that might be involved in regulating the migration of the miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced cells were analyzed. The levels of IL-2, GM-CSF, and IFN-γ secreted into the medium by the miR-200b/200a/429 cluster- and miR-141/200c cluster-transduced MDA-MB-231 cells after 48 h of culture were lower than those of the control MDA-MB-231 cells. However, the secreted VEGF-A levels were significantly higher in the conditioned medium of the miR-200b/200a/429 cluster-transduced cells (1.64 ± 0.03-fold) and miR-141/200c cluster-transduced cells (2.66 ± 0.09-fold) than in that of the control MDA-MB-231 cells (Fig. 3a, control vs. miR-200ab, p = 0.007 and control vs. miR-200c, p = 0.0007). Increased secretion of VEGF-A was higher in miR-141/200c cluster-transduced cells than miR-200b/200a/429 cluster-transduced cells (miR-200ab vs. miR-200c, p = 0.0001). The secreted levels of cytokines and growth factors were also evaluated in the miR-141/200c cluster-transduced HCC-38 and Hs578T cells. VEGF-A secretions were significantly increased in miR-141/200c cluster-transduced HCC-38 (1.75 ± 0.03-fold, p = 0.00001) and miR-141/200c cluster-transduced Hs578T cells (1.17 ± 0.16-fold, p = 0.004) relative to control cells (Additional file 4: Figure S4B, E). The effect of miR-141/200c overexpression on increased cell migration and VEGF-A secretion was greater in MDA-MB-231 cells relative to HCC-38 and Hs578T cells. There were no definite differences in VEGF-A secretion levels between the miR-141/200c cluster–transduced Hs578T and the control Hs578T cells. These results were explained by the phenotypic and functional heterogeneity among cancer cells in TNBC cells. The VEGF-A expression levels were also examined in the miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells, but the transduction of miR-200b/200a/429 cluster (1.18 ± 0.31-fold) or miR-141/200c cluster (0.90 ± 0.34-fold) did not affect VEGF-A expression (Fig. 3b, right, control vs. miR-200ab, p = 0.38 and control vs. miR-200c, p = 0.45). The miR-141/200c cluster–transduced HCC-38 cells (0.92 ± 0.09-fold) also expressed VEGF-A levels that were similar to the control HCC-38 cells (Additional file 6: Figure S6A, B, p = 0.46).
Blocking VEGF-A activity inhibited the enhanced migration and invasion of miR-200b/200a/429 cluster or miR-141/200c cluster-transduced MDA-MB-231 cells
To examine whether secreted VEGF-A regulates the migratory and invasive abilities of the miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells, trans-well migration and invasion assays were performed after treatment with anti-VEGF-A-neutralizing antibodies. The anti-VEGF-A-neutralizing antibodies were added to both the upper and lower chamber, which would allow their effects to last throughout the entire experimental period. Figure 3c, d d showed that the anti-VEGF-A-neutralizing antibodies completely inhibited the increased migratory (control vs. miR-200ab, p = 0.0001 and control vs. miR-200c, p = 0.002) and invasive (control vs. miR-200ab, p = 0.02 and control vs. miR-200c, p = 0.001) abilities of the miR-200b/200a/429 cluster- and miR-141/200c cluster-transduced cells. These data clearly indicated that secreted VEGF-A was involved in promoting the migration and invasion of both miR-200b/200a/429 cluster- and miR141/200c cluster-transduced cells. The administration of anti-VEGF-A-antibodies for neutralizing VEGF-A in cultured medium suppressed the migration increased by miR-141/200c overexpression in both the miR-141/200c cluster-transduced HCC-38 and miR-141/200c cluster-transduced Hs578T cells (Additional file 4: Figure S4C, F). The anti-VEGF-A-neutralizing antibodies also completely inhibited the increased migratory ability (control vs. miR-200c, p < 0.001) of the miR-141/200c cluster-transduced HCC-38 cells (Additional file 4: Figure S4C, untreated vs. VEGF-A Ab, p < 0.001). These results supported that VEGF-A secretion was associated with enhancing migration ability in TNBC cells. The anti-VEGF-A-neutralizing antibodies partly blocked the enhanced migratory ability (control vs. miR-200c, untreated, 1.76 ± 0.44-fold, p = 0.0003) of both the control (0.80 ± 0.07-fold, p = 0.003) and the miR-141/200c cluster-transduced Hs578T cells (1.37 ± 0.21-fold, p = 0.003), but the miR-141/200c cluster-transduced Hs578T cells still showed increased migratory ability compared with that of the control cells (control vs. miR-200c, VEGF-A Ab treated, p < 0.001, Additional file 4: Figure S4F). These results imply that other factors besides VEGF-A may also be involved in promoting migration in miR-141/200c cluster-transduced Hs578T cells.
The miR-141/200c cluster activated the FAK and PI3K/AKT signaling pathway by secreted VEGF-A, resulting in the promotion of the migratory and invasive abilities of MDA-MB-231 cells
To ascertain the VEGF-A-mediated intracellular signaling pathway responsible for the increased migratory and invasive abilities of miR-200b/200a/429 cluster- or miR141/200c cluster-transduced MDA-MB-231 cells, the levels of FAK and AKT phosphorylation were evaluated in cells treated with 5 μg/ml anti-VEGF-A-neutralizing antibodies. Treatment with anti-VEGF-A-neutralizing antibodies for both 30 min and 48 h remarkably decreased the level of FAK and AKT phosphorylation in the miR-141/200c cluster-transduced MDA-MB-231 cells and the control cells but did not affect the level of phosphorylated FAK and AKT in the miR-200b/200a/429 cluster-transduced MDA-MB-231 cells (Fig. 3e, f ). In contrast, the level of ERK phosphorylation was increased by anti-VEGF-A-neutralizing antibodies in miR-141/200c-transduced cells and control cells. These data implied that FAK and AKT acted as a direct link between elevated VEGF-A secretion and the consequential signal transduction related to enhanced migration of miR-141/200c cluster-transduced MDA-MB-231 cells, but the increased secretion of VEGF-A in miR-200b/200a/429 cluster-transduced MDA-MB-231 cells was not directly followed by activation of the FAK and AKT-mediated signaling pathway, which was possibly induced by the other signaling pathways.
Exogenous VEGF-A stimulated migration of MDA-MB-231 cells by activating the FAK and PI3K/AKT pathway
To confirm the role of VEGF-A as a migration-activating mediator in miR-200b/200a/429 cluster- or miR-141/200c cluster-transduced MDA-MB-231 cells, a trans-well migration assay was assessed in MCF-7 and MDA-MB-231 cells treated with exogenous VEGF-A protein. The representative crystal violet staining of exogenous VEGF-A (10 ng/ml)-treated MCF-7 and MDA-MB-231 cells that had migrated through the trans-well membranes after 48 h is shown in Fig. 4a. A quantitative analysis of a crystal violet assay showed that exogenous VEGF-A significantly increased MDA-MB-231 cell migration up to 1.50 ± 0.16-fold but had no effect on the migratory ability of MCF-7 cells compared with that of untreated cells (Fig. 4b, untreated vs. VEGF-A, p = 0.003). In addition, we investigated whether VEGF-A played a role as a migration-activating mediator in HCC-38 cells. As expected, VEGF-A treatment significantly increased HCC-38 cell migration up to 1.56 ± 0.26-fold (Additional file 7: Figure S7B, untreated vs. VEGF-A, p < 0.001).
To verify the exogenous VEGF-A-mediated intracellular signaling pathways responsible for the increased migratory ability of MDA-MB-231 cells, the levels of FAK, AKT, and ERK phosphorylation were evaluated. After cell starvation for 4 h, treatment with exogenous VEGF-A for 15 min, 30 min, 60 min, and 48 h induced the periodic fluctuation of the phosphorylation levels of FAK and AKT and generally increased phosphorylated FAK and AKT, but did not affect the phosphorylation level of ERK in MDA-MB-231 cells (Fig. 4c, d). These data indicated that the activation of the FAK and PI3K/AKT signaling pathways mediated by the exogenous VEGF-A increased the migratory ability of MDA-MB-231 cells. Similar results that VEGF-A stimulated FAK, AKT, and ERK were also observed in HCC-38 cells (Additional file 7: Figure S7C, D).