title: A Screen for Epigenetically Silenced microRNA Genes in Gastrointestinal Stromal Tumors.
abstract: BACKGROUND
  Dysregulation of microRNA (miRNA) has been implicated in gastrointestinal stromal tumors (GISTs) but the mechanism is not fully understood. In this study, we aimed to explore the involvement of epigenetic alteration of miRNA genes in GISTs.
  
  
  METHODS
  GIST-T1 cells were treated with 5-aza-2'-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which miRNA expression profiles were analyzed using TaqMan miRNA arrays. DNA methylation was then analyzed using bisulfite pyrosequencing. The functions of miRNAs were examined using MTT assays, wound-healing assays, Boyden chamber assays and Matrigel invasion assays. Gene expression microarrays were analyzed to assess effect of ectopic miRNA expression in GIST-T1 cells.
  
  
  RESULTS
  Of the 754 miRNAs analyzed, 61 were significantly upregulated in GIST-T1 cells treated with 5-aza-dC plus PBA. Among those, 21 miRNA genes were associated with an upstream CpG island (CGI), and the CGIs of miR-34a and miR-335 were frequently methylated in GIST-T1 cells and primary GIST specimens. Transfection of miR-34a or miR-335 mimic molecules into GIST-T1 cells suppressed cell proliferation, and miR-34a also inhibited migration and invasion by GIST-T1 cells. Moreover, miR-34a downregulated a number of predicted target genes, including PDGFRA. RNA interference-mediated knockdown of PDGFRA in GIST-T1 cells suppressed cell proliferation, suggesting the tumor suppressive effect of miR-34a is mediated, at least in part, through targeting PDGFRA.
  
  
  CONCLUSIONS
  Our results suggest that miR-34a and miR-335 are candidate tumor suppressive miRNAs in GISTs, and that they are frequent targets of epigenetic silencing in GISTs.
authors.affiliation: Department of Gastroenterology, Rheumatology and Clinical Immunology, Sapporo Medical University School of Medicine, Sapporo, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Isosaka
authors.affiliation: Department of Molecular Biology, Sapporo Medical University, Sapporo, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Niinuma
authors.affiliation: Center for Translational Research, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Nojima
authors.affiliation: Department of Molecular Biology, Sapporo Medical University, Sapporo, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kai
authors.affiliation: Department of Gastroenterology, Rheumatology and Clinical Immunology, Sapporo Medical University School of Medicine, Sapporo, Japan; Department of Molecular Biology, Sapporo Medical University, Sapporo, Japan.
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Yamamoto
authors.affiliation: Department of Gastroenterology, Rheumatology and Clinical Immunology, Sapporo Medical University School of Medicine, Sapporo, Japan.
authors.forename: Reo
authors.initials: R
authors.lastname: Maruyama
authors.affiliation: Department of Surgery, Surgical Oncology and Science, Sapporo Medical University School of Medicine, Sapporo, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nobuoka
authors.affiliation: National Cancer Center Hospital East, Kashiwa, Japan.
authors.forename: Toshirou
authors.initials: T
authors.lastname: Nishida
authors.affiliation: Department of Surgery, Sanjo General Hospital, Sanjo City, Niigata, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kanda
authors.affiliation: Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Taguchi
authors.affiliation: Department of Surgical Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Hasegawa
authors.affiliation: Medical Genome Science, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tokino
authors.affiliation: Department of Surgery, Surgical Oncology and Science, Sapporo Medical University School of Medicine, Sapporo, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Hirata
authors.affiliation: Department of Molecular Biology, Sapporo Medical University, Sapporo, Japan.
authors.forename: Hiromu
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Gastroenterology, Rheumatology and Clinical Immunology, Sapporo Medical University School of Medicine, Sapporo, Japan.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Shinomura
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D018899:CpG Islands; D000077209:Decitabine; D005770:Gastrointestinal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D046152:Gastrointestinal Stromal Tumors / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D010654:Phenylbutyrates / Q000494:pharmacology; D012334:RNA, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000096:biosynthesis / Q000235:genetics
chemical_list: C556347:MIRN335 microRNA, human; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D009363:Neoplasm Proteins; D010654:Phenylbutyrates; D012334:RNA, Neoplasm; D000077209:Decitabine; C075773:4-phenylbutyric acid; D020797:Receptor, Platelet-Derived Growth Factor beta; D001374:Azacitidine