title: Serial selection for invasiveness increases expression of miR-143/miR-145 in glioblastoma cell lines.
abstract: BACKGROUND
  Glioblastoma multiforme (GBM) is the most common primary central nervous system malignancy and its unique invasiveness renders it difficult to treat. This invasive phenotype, like other cellular processes, may be controlled in part by microRNAs - a class of small non-coding RNAs that act by altering the expression of targeted messenger RNAs. In this report, we demonstrate a straightforward method for creating invasive subpopulations of glioblastoma cells (IM3 cells). To understand the correlation between the expression of miRNAs and the invasion, we fully profiled 1263 miRNAs on six different cell lines and two miRNAs, miR-143 and miR-145, were selected for validation of their biological properties contributing to invasion. Further, we investigated an ensemble effect of both miR-143 and miR-145 in promoting invasion.
  
  
  METHODS
  By repeated serial invasion through Matrigel®-coated membranes, we isolated highly invasive subpopulations of glioma cell lines. Phenotypic characterization of these cells included in vitro assays for proliferation, attachment, and invasion. Micro-RNA expression was compared using miRCURY arrays (Exiqon). In situ hybridization allowed visualization of the regional expression of miR-143 and miR-145 in tumor samples, and antisense probes were used investigate in vitro phenotypic changes seen with knockdown in their expression.
  
  
  RESULTS
  The phenotype we created in these selected cells proved stable over multiple passages, and their microRNA expression profiles were measurably different. We found that two specific microRNAs expressed from the same genetic locus, miR-143 and miR-145, were over-expressed in our invasive subpopulations. Further, we also found that combinatorial treatment of these cells with both antisense-miRNAs (antimiR-143 and -145) will abrogated their invasion without decreasing cell attachment or proliferation.
  
  
  CONCLUSIONS
  To best of our knowledge, these data demonstrate for the first time that miR-143 and miR-145 regulate the invasion of glioblastoma and that miR-143 and -145 could be potential therapeutic target for anti-invasion therapies of glioblastoma patients.
authors.affiliation: Department of Neuroscience and Experimental Therapeutics, Texas A&M Health Science Center College of Medicine, Bryan, TX 77843, USA. swkoo@tamu.edu
authors.forename: Sunwoo
authors.initials: S
authors.lastname: Koo
authors.forename: Gail S
authors.initials: GS
authors.lastname: Martin
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Schulz
authors.forename: Matthew
authors.initials: M
authors.lastname: Ronck
authors.forename: L Gerard
authors.initials: LG
authors.lastname: Toussaint
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016543:Central Nervous System Neoplasms / Q000378:metabolism* / Q000473:pathology; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D016372:RNA, Antisense / Q000494:pharmacology; D051381:Rats
chemical_list: C542359:MIRN143 microRNA, human; C541010:MIRN145 microRNA, human; D035683:MicroRNAs; D016372:RNA, Antisense