title: 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.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Radiology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea.|Department of Biomedical Science, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, Korea.
authors.forename: Sul Ki
authors.initials: SK
authors.lastname: Choi
authors.affiliation: Department of Radiology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea.
authors.forename: Hoe Suk
authors.initials: HS
authors.lastname: Kim
authors.affiliation: Department of Radiology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea.
authors.forename: Tiefeng
authors.initials: T
authors.lastname: Jin
authors.affiliation: Department of Radiology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea.
authors.forename: Eun Hye
authors.initials: EH
authors.lastname: Hwang
authors.affiliation: Department of Radiology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea.
authors.forename: Minji
authors.initials: M
authors.lastname: Jung
authors.affiliation: Department of Radiology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea. moonwk@snu.ac.kr.|Department of Biomedical Science, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, Korea. moonwk@snu.ac.kr.
authors.forename: Woo Kyung
authors.initials: WK
authors.lastname: Moon
mesh_terms: D002460:Cell Line; D002465:Cell Movement; D049109:Cell Proliferation; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D005810:Multigene Family; D009361:Neoplasm Invasiveness; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D064726:Triple Negative Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: C533895:MIRN141 microRNA, human; C533896:MIRN200 microRNA, human; D035683:MicroRNAs; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019869:Phosphatidylinositol 3-Kinases; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D051057:Proto-Oncogene Proteins c-akt
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)