title: MicroRNA down-regulated in human cholangiocarcinoma control cell cycle through multiple targets involved in the G1/S checkpoint.
abstract: MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study we aimed at elucidating regulatory pathways and mechanisms through which miR-494, one of the miR species found to be down-regulated in cholangiocarcinoma (CCA), participates in cancer homeostasis. miR-494 was identified as down-regulated in CCA based on miR arrays. Its expression was verified with quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). To enforce miR expression, we employed both transfection methods, as well as a retroviral construct to stably overexpress miR-494. Up-regulation of miR-494 in cancer cells decreased growth, consistent with a functional role. mRNA arrays of cells treated with miR-494, followed by pathway analysis, suggested that miR-494 impacts cell cycle regulation. Cell cycle analyses demonstrated that miR-494 induces a significant G1/S checkpoint reinforcement. Further analyses demonstrated that miR-494 down-regulates multiple molecules involved in this transition checkpoint. Luciferase reporter assays demonstrated a direct interaction between miR-494 and the 3'-untranslated region of cyclin-dependent kinase 6 (CDK6). Last, xenograft experiments demonstrated that miR-494 induces a significant cancer growth retardation in vivo.
  
  
  CONCLUSIONS
  Our findings demonstrate that miR-494 is down-regulated in CCA and that its up-regulation induces cancer cell growth retardation through multiple targets involved in the G1-S transition. These findings support the paradigm that miRs are salient cellular signaling pathway modulators, and thus represent attractive therapeutic targets. miR-494 emerges as an important regulator of CCA growth and its further study may lead to the development of novel therapeutics.
authors.affiliation: Division of Gastroenterology and Hepatology, Johns Hopkins Hospital, Baltimore, MD 21205, USA.
authors.forename: Alexandru V
authors.initials: AV
authors.lastname: Olaru
authors.forename: Gabriel
authors.initials: G
authors.lastname: Ghiaur
authors.forename: Sumitaka
authors.initials: S
authors.lastname: Yamanaka
authors.forename: Delgermaa
authors.initials: D
authors.lastname: Luvsanjav
authors.forename: Fangmei
authors.initials: F
authors.lastname: An
authors.forename: Irinel
authors.initials: I
authors.lastname: Popescu
authors.forename: Sorin
authors.initials: S
authors.lastname: Alexandrescu
authors.forename: Sarah
authors.initials: S
authors.lastname: Allen
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Pawlik
authors.forename: Michael
authors.initials: M
authors.lastname: Torbenson
authors.forename: Christos
authors.initials: C
authors.lastname: Georgiades
authors.forename: Lewis R
authors.initials: LR
authors.lastname: Roberts
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Gores
authors.forename: Anne
authors.initials: A
authors.lastname: Ferguson-Smith
authors.forename: Maria I
authors.initials: MI
authors.lastname: Almeida
authors.forename: George A
authors.initials: GA
authors.lastname: Calin
authors.forename: Esteban
authors.initials: E
authors.lastname: Mezey
authors.forename: Florin M
authors.initials: FM
authors.lastname: Selaru
mesh_terms: D000818:Animals; D001650:Bile Duct Neoplasms / Q000235:genetics* / Q000378:metabolism; D001653:Bile Ducts, Intrahepatic / Q000378:metabolism*; D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics*; D018281:Cholangiocarcinoma / Q000235:genetics* / Q000378:metabolism; D051361:Cyclin-Dependent Kinase 6 / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D009368:Neoplasm Transplantation; D014162:Transfection; D014183:Transplantation, Heterologous
chemical_list: C547168:MIRN494 microRNA, human; D035683:MicroRNAs; D051361:Cyclin-Dependent Kinase 6