title: Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer.
abstract: MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.
authors.affiliation: Colorectal Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Lise Lotte
authors.initials: LL
authors.lastname: Christensen
authors.affiliation: Clinical Biochemistry, Glostrup Research Institute, Glostrup Hospital, Glostrup, Denmark; Molecular Sleep Laboratory, Department of Diagnostics, and Danish Center for Sleep Medicine, Department of Clinical Neurophysiology, Glostrup Hospital, University of Copenhagen, Glostrup, Denmark.
authors.forename: Anja
authors.initials: A
authors.lastname: Holm
authors.affiliation: Medical Biotechnology, VTT Technical Research Centre of Finland, Turku, Finland.
authors.forename: Juha
authors.initials: J
authors.lastname: Rantala
authors.affiliation: Medical Biotechnology, VTT Technical Research Centre of Finland, Turku, Finland.
authors.forename: Olli
authors.initials: O
authors.lastname: Kallioniemi
authors.affiliation: Colorectal Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Mads H
authors.initials: MH
authors.lastname: Rasmussen
authors.affiliation: Bladder Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Marie S
authors.initials: MS
authors.lastname: Ostenfeld
authors.affiliation: Department of Biomedicine, University of Aarhus, Aarhus, Denmark.
authors.forename: Frederik
authors.initials: F
authors.lastname: Dagnaes-Hansen
authors.affiliation: Colorectal Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Bodil
authors.initials: B
authors.lastname: Øster
authors.affiliation: Colorectal Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Troels
authors.initials: T
authors.lastname: Schepeler
authors.affiliation: Colorectal Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Heidi
authors.initials: H
authors.lastname: Tobiasen
authors.affiliation: Colorectal Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Kasper
authors.initials: K
authors.lastname: Thorsen
authors.affiliation: Ludwig Colon Cancer Initiative Laboratory, Ludwig Institute for Cancer Research, Melbourne, Australia; Faculty of Medicine, Dentistry and Health Sciences, Department of Surgery, University of Melbourne, Melbourne, Australia.
authors.forename: Oliver M
authors.initials: OM
authors.lastname: Sieber
authors.affiliation: Ludwig Colon Cancer Initiative Laboratory, Ludwig Institute for Cancer Research, Melbourne, Australia; Faculty of Medicine, Dentistry and Health Sciences, Department of Surgery, University of Melbourne, Melbourne, Australia; Department of Medical Oncology, Royal Melbourne and Western Hospital, Melbourne, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Gibbs
authors.affiliation: Colorectal Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Philippe
authors.initials: P
authors.lastname: Lamy
authors.affiliation: Department of Oncology, Vejle Hospital, University of Southern Denmark, Vejle, Denmark.
authors.forename: Torben F
authors.initials: TF
authors.lastname: Hansen
authors.affiliation: Department of Oncology, Vejle Hospital, University of Southern Denmark, Vejle, Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Jakobsen
authors.affiliation: Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Riising
authors.affiliation: Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kristian
authors.initials: K
authors.lastname: Helin
authors.affiliation: Department of Pathology and Genetics, Pomeranian Medical University, Szczecin, Poland.
authors.forename: Jan
authors.initials: J
authors.lastname: Lubinski
authors.affiliation: Department of Pathology, Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Rikke
authors.initials: R
authors.lastname: Hagemann-Madsen
authors.affiliation: Department of Surgery, Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Laurberg
authors.affiliation: Colorectal Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Torben F
authors.initials: TF
authors.lastname: Ørntoft
authors.affiliation: Colorectal Cancer Research Group, Department of Molecular Medicine (MOMA), Aarhus University Hospital, University of Aarhus, Aarhus, Denmark.
authors.forename: Claus L
authors.initials: CL
authors.lastname: Andersen
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology*; D019175:DNA Methylation / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D045325:HCT116 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007413:Intestinal Mucosa / Q000473:pathology; D061346:Laser Capture Microdissection; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism; D009367:Neoplasm Staging; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D035683:MicroRNAs; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering