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BMC Cancer. 2016 Aug 2;16:570. doi: 10.1186/s12885-016-2620-7.

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.

Author information

1
Department of Radiology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea.
2
Department of Biomedical Science, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, Korea.
3
Department of Radiology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea. moonwk@snu.ac.kr.
4
Department of Biomedical Science, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, Korea. moonwk@snu.ac.kr.

Abstract

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.

KEYWORDS:

Focal adhesion kinase (FAK); Invasion; Migration; Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K); Protein kinase B (AKT); Triple-negative breast cancer (TNBC); Vascular endothelial growth factor (VEGF); microrna-200 (miR-200)

PMID:
27484639
PMCID:
PMC4969651
DOI:
10.1186/s12885-016-2620-7
[Indexed for MEDLINE]
Free PMC Article

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