Overexpression of the miR-141/200c cluster promotes the migratory and invasive ability of triple-negative breast cancer cells through the activation of the FAK and PI3K/AKT signaling pathways by secreting VEGF-A

Background The role of microRNA-200 (miR-200) family members in the migration and invasion of breast cancer is controversial. This study investigated the mechanisms by which the miR-200 family members modulated the migratory and invasive abilities of an aggressive triple-negative breast cancer (TNBC) cell line, MDA-MB-231. Methods The miR-200 family (miR-200b/200a/429 and miR-141/200c clusters) and green fluorescence protein (GFP) were transduced into MDA-MB-231 cells using a lentiviral system. Stable cells highly expressing the miR-200 family and GFP were isolated by puromycin selection and fluorescence-activated cell sorting. Gene expression was evaluated using real-time polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR). The migratory and invasive abilities were assessed using trans-well and wound-healing assays. The secreted cytokines and growth factors in cultured media were quantified using a Bio-Plex200 multiplex array system. Western blot assays and immunofluorescence staining were conducted to investigate miR-200 family-regulated signaling pathways. The entire dataset obtained in this study was statistically evaluated using a one-way ANOVA followed by a t-test. Results The stable overexpression of the miR-200b/200a/429 or miR-141/200c cluster suppressed cell growth and significantly increased migration and invasion of MDA-MB-231 cells. miR-141/200c overexpression was more effective in decreasing cell growth and promoting migration and invasion of MDA-MB-231 cells than was miR-200b/200a/429 overexpression. In addition, the overexpression of the miR-200b/200a/429 or miR-141/200c cluster led to an increase in the phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT). Chemical inhibitors of FAK and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT suppressed the migration and invasion of MDA-MB-231 cells that was enhanced by the overexpression of the miR-200b/200a/429 or miR-141/200c cluster. Compared to the miR-200b/200a/429 cluster-transduced MDA-MB-231 cells, the miR-141/200c cluster-transduced MDA-MB-231 cells exhibited a significant increase in vascular endothelial growth factor (VEGF)-A secretion and integrin-alphaV (integrin-αV) expression. Treatment with an anti-VEGF-A-neutralizing antibody inhibited the increase in migration and invasion in both the miR-200b/200a/429- and miR-141/200c-transduced MDA-MB-231 cells but significantly reduced the phosphorylation of FAK and AKT in only the miR-141/200c cluster-transduced MDA-MB-231 cells. Conclusions Taken together, our data demonstrate a mechanism in which the miR-141/200c cluster, through FAK- and PI3K/AKT-mediated signaling by means of increased VEGF-A secretion, promotes the migratory and invasive abilities of MDA-MB-231 cells. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2620-7) contains supplementary material, which is available to authorized users.


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Conclusions: Taken together, our data demonstrate a mechanism in which the miR-141/200c cluster, through FAKand PI3K/AKT-mediated signaling by means of increased VEGF-A secretion, promotes the migratory and invasive abilities of MDA-MB-231 cells.

Background
Aberrant expression of microRNAs (miRs), which are small non-coding RNA molecules consisting of approximately 22 nucleotides, has been identified in human cancer, where the miRNA signature is associated with specific clinical and biological features [1]. The micro-RNAs related to cancers may act as tumor suppressors or oncogenes, depending on the cancer type [2,3]. The miR-200 family member genes are clustered at two locations in the genome: the miR-200b/200a/429 cluster and the miR-141/200c cluster [4]. The miR-200 family members repress the epithelial-to-mesenchymal transition (EMT), cancer cell migration, tumor growth, and metastasis by directly targeting specific genes, such as ZEB1, Suz12, moesin, and AP-2γ [4,5]. In contrast, the miR-200 family members have been shown to enhance the migration ability of breast cancer cells and to promote the metastatic colonization of breast cancer cells through up-regulating the expression of E-cadherin and downregulating that of ZEB2 and Sec23a [6,7]. In a recent study, high expression of the miR-200 family was associated with a high probability of relapse, poor survival, and distant metastasis in breast cancer patients [8]. The loss of miR-200c expression has also been related to the induction of an aggressive, invasive, and chemoresistant phenotype of non-small cell lung cancer [9]. Conflicting results have been obtained in studies of the role of each miR-200 family member in repressing or enhancing cancer cell migration and invasion as well as the tumor growth and metastasis of diverse cancers, including breast cancer [10,11].
Triple-negative breast cancer (TNBC) lacking estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression, is a highly invasive and metastatic form of breast cancer with a generally poorer prognosis than that of other breast cancer subtypes [12]. It is important to develop new treatment strategies based on a better understanding of the underlying mechanisms regulating the aggressive behavior of TNBCs. TNBCs express the miR-200 family members at a significantly lower level than do other subtypes of breast cancer, such as ER-positive or HER2-positive breast cancer [13]. Only a small number of the miR-200 target genes that are involved in breast cancer cell migration and metastasis have been identified [4][5][6], and few studies of the role of the miR-200b/200a/ 429 or miR-141/200c cluster in human TNBC have been conducted. The biological relevance of the function of the miR-200b/200a/429 or miR-141/200c cluster in human TNBC remains to be discovered.
Synthetic miR-200b directly downregulates vascular endothelial growth factor (VEGF) in endothelial cells and prevents the diabetes-induced increase in VEGF, thus inhibiting angiogenesis in diabetic retinopathy [14]. Chemokine CCL5 (formerly RANTES) of the CCchemokine family, which plays a critical role in local invasion and distant metastasis in chondrosarcoma, promotes VEGF expression and angiogenesis by downregulating miR-200b [15]. The miR-200c radiosensitized the lung cancer cell line, A549 by targeting the VEGF-VEGFR2 pathway [16]. From these reports, we speculated that the overexpression of the miR-200 family can regulate the expression and secretion of cytokines and growth factors involving in cell growth and migratory and invasive abilities of TNBC cells.
In the present study, we used MDA-MB-231 cells, a typical human TNBC cell line, which were stably transduced with lentivirus carrying miR-200 family. We found that the overexpression of the miR-141/200c cluster promoted stronger migration and invasion as well as higher VEGF-A secretion in MDA-MB-231 cells. Therefore, we investigated, in detail, the mechanisms by which two miR-200 family members, the miR-200b/200a/429 cluster and the miR-141/200c cluster, regulated MDA-MB-231 cell migration and invasion. We demonstrate that the overexpression of the miR-141/200c cluster in MDA-MB-231 cells increased VEGF-A secretion, which enhanced the migratory ability of the cells through the activation of focal adhesion kinase (FAK) and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/ protein kinase B (AKT) signaling pathway.

Lentiviral transduction
Viral vectors containing either the miR-200b/200a/429 cluster (GenBank ID: 406984 406983) or the miR-141/ 200c cluster (GenBank ID: 406985 406933) constructs and the green fluorescent protein (GFP) construct were kindly supplied by Dr. Gregory J. Goodall of the University of Adelaide (Adelaide, Australia). A viral vector (pLenti M1.41) containing GFP was used as a control vector. Lentiviral transduction was conducted according to the manufacturer's instructions. Briefly, cells were seeded at a density of approximately 10-25 % confluency (1 × 10 5 cells) in 6-well plates and were maintained at 37°C with 5 % CO 2 . Following an overnight culture, the culture medium was removed. Aliquots of the lentiviral stocks containing the miR-200b/200a/429 cluster or the miR-141/200c cluster construct as well as the control virus were gently mixed with 8 μg/ml polybrene and added to each well. After 6 h of transduction, the medium was replaced with fresh complete medium. Transduced cells with a cell density of greater than 90 % confluency were selected using medium containing 3 μg/ml puromycin for 2 weeks. Then, the GFP-positive cells were sorted from the selected cells using a FACS-Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cancer cells stably expressing the miR-200b/200a/429 cluster or the miR-141/200c cluster and GFP, denoted as miR-200ab cells and miR-200c cells, respectively, and cancer cells containing the pLenti M1.41 vector, denoted as control cells, were generated and expanded for use in all subsequent studies.

Quantitative real-time PCR
TaqMan MicroRNA Assays (Applied Biosystems, South San Francisco, CA, USA) were used to quantify the levels of mature miRNAs, following the manufacturer's instructions. The miRNAs were isolated from cells using the mirVana miRNA isolation kit (Applied Biosystems), and the specific primers for detecting miR-200a, miR-200b, and, miR-200c were purchased from Applied Biosystems. Reverse transcription was performed using the TaqMan microRNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions. The traditional TaqMan Assay control, 18 s rRNA, was used as the endogenous control. Each TaqMan Assay was conducted in triplicate.

Cell viability and proliferation assay
In vitro cell viability and proliferation were assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, 5 × 10 3 cells were allowed to adhere in a high-humidity environment in 5 % CO 2 at 37°C in 96-well culture plates. At 1 d, 3 d, and 5 d after cell seeding, the MTT solution (a final concentration of 1 mg/ml) was added, and the cells were incubated for 1 h. At the end of the incubation period, the MTT solution was carefully removed, and 150 μl of dimethyl sulfoxide was added to each well. The plates were maintained on a rocker shaker for 10 min at 25°C, and then the amount of MTT formazan crystals formed by the viable cells was determined using a spectrophotometer based on the absorbance at 540 nm (GE Healthcare, Piscataway, NJ, USA). Flow cytometry analysis using 7-AAD was performed to confirm cell viability.

Migration and invasion assay
To assess the cell migratory ability, 2-5 × 10 4 cells were suspended in 100 μl of medium with or without 10 % FBS and deposited in the upper chambers of a trans-well plate with 8.0-μm pores (BD Biosciences) with a noncoated membrane. For the invasion assays, 5 × 10 4 cells were plated in 2 % Matrigel™ (BD Biosciences) basement membrane matrix-coated upper chambers in a transwell plate with 8.0-μm pores. The lower chambers were filled with 600 μl of medium supplemented with 10 % FBS, and the cells were incubated for 48 h at 37°C in the presence or absence of an FAK inhibitor (5 μM PF573228, Sigma, St. Louis, MO, USA), a PI3K/AKT inhibitor (20 μM LY294002, Cell Signaling Technology, Danvers, MA, USA), 5 μg/ml VEGF-neutralizing antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or 10 ng/ml VEGF-A protein (Sigma). Each inhibitor, VEGF-neutralizing antibody, or VEGF-A were added in both the upper and lower chambers. No chemoattractants were used in the lower chamber for either the migration or invasion assays. The cells that migrated from the upper chamber were stained using a crystal violet solution (0.5 % crystal violet in 20 % methanol) for 5 min. Unbound crystal violet was removed by rinsing using distilled water. The cells were subsequently air-dried, and the crystal violet was eluted from the cells using a solution of 1 % sodium dodecyl sulfate (SDS). The absorbance of crystal violet at 550 nm was measured using a spectrophotometer (GE Healthcare).

Wound-healing assay
Cells were seeded at 5 × 10 4 cells per well in six-well plates and cultured under permissive conditions until reaching 90 % confluence. After 24 h, each confluent cell monolayer was lightly and quickly scratched using a sterile plastic tip to produce a straight line. The debris was removed, and the edge of the scratch was smoothed by washing with PBS. The cells were cultured for 6 h in complete medium, after which, the lateral migratory activity was evaluated based on the area occupied by the cells that had entered the scratch line at 0 h. Images were acquired using a microscope (Leica, Wetzlar, Germany) equipped with a CCD camera (Leica). The migration rates were calculated according to the equation percentage wound healing = {(wound length at 0 h) -(wound length at 6 h)} / (wound length at 0 h) × 100. The mean results of three straight distances (upper edge, middle, and lower edge) in scratch area were evaluated as wound lengths. Length quantification was performed using ImageJ software (NIH, Bethesda, MD, USA).

Measurement of cytokines and growth factors
Samples of 1-2 × 10 5 cells were seeded in 6-well plates. After a two-day culture period, when cells were at 90 % confluency, the conditioned medium was harvested. The levels of secreted cytokines and growth factor (IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-CSF, IFN-γ, and VEGF-A) were quantified using the Bio-Plex200 multiplex array system according to the recommended protocol (Bio-Rad). All samples and standardized solutions were analyzed in triplicate.

Western blotting
The cells were lysed in RIPA buffer (Sigma). The proteins were separated using SDS-polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes. The membranes were blocked using 5 % skim milk in Tris-buffered saline containing Tween and incubated with primary antibodies directed against ERK, phospho-ERK, AKT (anti-rabbit polyclonal antibody, Cell Signaling Technology), phospho-AKT (anti-mouse polyclonal antibody, Cell Signaling Technology), FAK (anti-rabbit polyclonal antibody, Invitrogen), phospho-FAK (anti-rabbit polyclonal antibody, Invitrogen), integrin-αV (anti-rabbit polyclonal antibody, Santa Cruz Biotechnology) or β-actin (anti-mouse polyclonal antibody, Sigma) overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). The blotted membranes were visualized using enhanced chemiluminescence reagents (GE Healthcare). Western blot quantification was performed using ImageJ software.

Statistical analyses
For the entire dataset obtained in this study, the mean values ± standard deviations were calculated from the results of at least three independent experiments and were statistically evaluated using a one-way ANOVA followed by the t-test. For all tests, p-values of less than 0.05 were considered significant. 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 clusteror 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.

Overexpression
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 clusteror 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 (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  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.   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  (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).

Discussion
The role of miR-200 family in regulating the migration and invasion of different cancer cell types is controversial [10,11]. Moreover, only a small number of miR-200 target genes that regulate cell migration and cancer metastasis has been identified [4,6],  [17]. ZEB1 and SIP1 have been found to repress primary transcript and mature miR-200 expression in mesenchymal types of breast cancer cells, suggesting a downregulation of miR-200 in TNBC cells through a potential doublenegative feedback loop between ZEB1/SIP1 and the miR-200 family [18]. Manav K et al. reported the highest expression of miR-200s in the highly metastatic 4T1 cells, a mouse TNBC cell line, which was consistent with acquisition of epithelial traits in 4T1 cells compared with the weakly metastatic 4TO7 cells. In addition, 4T1 tumors exhibit spontaneous metastasis and colonization of distant organs, which is enhanced by miR-200 overexpression in experimental animal models, furthermore, higher expression of miR-200 levels were found in lungpleural metastasis samples relative to primary tumor samples in breast cancer patients. These data support the potential role of miR-200s in migration, invasion, metastatic colonization, and metastatic dissemination [6]. Recently, Avery-Kiejda KA et al. found that the miR-200 cluster is upregulated in invasive ductal carcinomas with both lymph node-positive and lymph node-negative TNBC compared with matched normal adjacent tissues [19]. Their reports of in vivo experimental and clinical evidence may indicate that tumor cell populations with increased aggressiveness may have higher miR-200 cluster levels than their less aggressive counterparts within the same TNBC and in normal tissues, the miR-200 cluster, though being generally reduced in TNBC compared with other subtypes, is upregulated in TNBC cells that may support metastatic dissemination. We showed here that the overexpression of miR-200b/200a/429 cluster or miR-141/200c cluster strongly promoted the migration and invasion of MDA-MB-231, HCC-38, and Hs578T cells, typical claudin-low and mesenchymal subtypes of TNBC cell lines [20], compared with those of an ER-positive breast cancer cell line, MCF-7 cells. The migratory and invasive ability of MDA-MB-231 cells was substantially more enhanced in those that overexpressed the miR-141/200c cluster than in those with the miR-200b/200a/429 cluster. These data suggest that the different roles of the miR-200 family members, such as miR-200a, miR-200b, miR-200c, miR-141, and miR-429, on the migration and invasion of different subtypes of human breast cancer cell lines classified by molecular characterization should be further investigated [21].
Dysregulation of the PI3K/AKT signaling pathway has been implicated in mammary carcinogenesis and was suggested to be the mechanism underlying the survival of invasive breast cancer cells [22]. Furthermore, an activated AKT signaling pathway, a common dysregulation observed in breast cancers, has been shown to promote cancer cell growth, survival, and metastasis [23]. FAK is a cytoplasmic tyrosine kinase that plays crucial roles in integrin-mediated signal transduction, and FAK localizes to the sites where transmembrane integrin receptors are clustered to mediate various intracellular signaltransduction pathways [5,24]. Many recent studies have reported that an increased level of FAK expression highly correlates with the invasiveness and metastasis of human tumors [25][26][27]. We found that the phosphorylation levels The regulation between the miR-200 family and FAK is not fully understood. A model indicating that a stiffer matrix of breast cancers will activate FAK, which inhibits the miR-200 family and allow for a mesenchymal phenotype has been proposed [28]. This model is not consistent with our observation that the overexpression of the miR-141/200c cluster or the miR-200b/200a/429 cluster increased clustering and expression of integrins and activated FAK and AKT, which regulate cell migration. Our finding proposes a positive cross-talk between FAK and overexpressed miR-200 in TNBC cells.
Many studies have been aimed at understanding the role of cytokines and growth factors in breast cancer progression. Some cytokines and growth factors (IL-1, IL-6, IL-11, TGF-β, and VEGF) stimulate the proliferation and invasion of breast cancer cells, whereas others (IL-12, IL-18, and IFN) suppress breast cancer progression [29]. A recent study reported that miR-200 inhibits angiogenesis by targeting IL-8 and CXCL1 secreted by tumor endothelial and cancer cells [30]. Our study demonstrated that the overexpression of the miR-200b/200a/ 429 cluster or the miR-141/200c cluster in MDA-MB-231 cells led to a decrease in the secretion of IL-2, IL-4, IL-5, IL-10, IL-13, GM-CSF, INF-γ and TNF-α but a significant increase in the secretion of VEGF-A. VEGF has been reported to activate the PI3K/AKT/forkhead signaling pathway to promote angiogenesis in human endothelial cells [31]. In addition, VEGF-A, the most potent angiogenic factor in tumor angiogenesis, induces oligodendrocyte precursor cell migration through a ROS-and FAK-dependent mechanism [32]. VEGF-A is known to be a direct target of miR-200b [33], but in our results, a significant decrease in VEGF-A levels assessed by western blotting was not observed in miR-200b/200a/429and miR-141/200c-transduced MDA-MB-231 cells or in miR-141/200c-transduced HCC-38 cells relative to control. Moreover, VEGF-A secretions in MDA-MB-231, HCC-38, and Hs578T cells were increased by the overexpression of the miR-141/200c cluster. The comprehensive interactions between miRNAs and transcription factors (TFs) are expected to comprise "wired" genetic networks to regulate the expression of target genes [34]. In examples of an incoherent feed-forward loop, the direct regulatory effect of TFs on the target gene (VEGF-A) is opposed to the indirect regulatory effect through miR-200 regulation. Taking the comprehensive interactions between miRNAs and protein-coding genes, we propose that miR-200 overexpression in TNBC cells can affect TFs (HIF-1, CREB) or signals (PI3K/AKT) to regulate VEGF-A secretion through an incoherent feed-forward loop. An autocrine loop for VEGF-A to induce breast cancer cell migration/invasion has been well documented [35,36]. From these reports, we assume that the VEGF-A secreted by miR-200 overexpression interacts with its receptors, such as neuropilin-1 (NP-1) and VEGFR, and stimulates the PI3K/AKT signaling pathway, thus promoting TNBC cell migration and invasion.
In this study, we demonstrated that treatment with VEGF-A led to an increase in migratory ability and activated FAK and the PI3K/AKT signaling pathway in MDA-MB-231 cells and HCC-38 cells. Our results strongly support that VEGF-A-mediated FAK or PI3K/ AKT signaling pathway modulates cancer cell migration and invasion. By down-regulating miR-200b expression through the PI3K/AKT signaling pathway, the chemokine CCL5 (formerly RANTES) promotes VEGF-dependent angiogenesis in human chondrosarcomas [15]. In addition, synthetic miR-200c downregulates VEGF-A by the direct targeting of the 3'UTR of VEGF-A mRNA in a lung cancer cell line [16]. Contrary to above studies, in this study, the stable overexpression of the miR-

Conclusions
The stable overexpression of the miR-141/200c cluster promoted the migratory and invasive ability of TNBC cells through the strong activation of FAK and the PI3K/ AKT signaling pathway by increasing VEGF-A secretion compared with the miR-200b/200a/429 cluster.