title: Claudin-1 is a novel target of miR-375 in non-small-cell lung cancer.
abstract: OBJECTIVE
  We previously reported low expression of miR-375 in squamous-cell carcinoma (SCC) and high expression in adenocarcinoma (AC) of the lung. miR-375's target genes and its function in non-small-cell lung cancer (NSCLC) have not been elucidated. Therefore, the present study was designed to identify the targets of miR-375 and to characterize its function in NSCLC.
  
  
  METHODS
  Candidate targets of miR-375 were determined using a prediction database and previous data on differential gene expression between SCC and AC. We evaluated miR-375 and target-gene expression levels in 12 NSCLC cell lines. The effect of miR-375 overexpression and knockdown was evaluated in NSCLC cell lines by transfecting them with an miR-375 precursor or inhibitor. A luciferase-reporter assay was performed to confirm a direct interaction between miR-375 and its target gene. Further, a wound-healing assay was performed to evaluate the effect of miR-375 overexpression on the migration of SK-MES-1 cells. Finally, to assess the clinical relevance, 63 clinical NSCLC samples were analyzed.
  
  
  RESULTS
  Claudin-1 (CLDN1) has 4 putative miR-375 target sites in its 3'-untranslated region, and this gene was determined to be a target of miR-375. CLDN1 messenger RNA and protein expression were attenuated by overexpression of miR-375 and increased by knockdown of miR-375 in NSCLC cell lines. In a luciferase-reporter assay, miR-375 overexpression resulted in a 3-fold repression of luciferase activity (P<0.001). Cell migration was promoted by miR-375 overexpression, suggesting a high potential for invasion and metastasis in NSCLC expressing high levels of miR-375. In clinical NSCLC samples, there was a negative correlation between miR-375 and CLDN1 expression (r=-0.35, P=0.005). In addition, high miR-375 expression was correlated with a shorter survival time among the clinical samples (P=0.043).
  
  
  CONCLUSIONS
  CLDN1 is a novel target of miR-375, and high miR-375 expression shortens survival in NSCLC.
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yoda
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Electronic address: ksoejima@cpnet.med.keio.ac.jp.
authors.forename: Kenzo
authors.initials: K
authors.lastname: Soejima
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Hamamoto
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yasuda
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Sohei
authors.initials: S
authors.lastname: Nakayama
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Satomi
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Terai
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Shinnosuke
authors.initials: S
authors.lastname: Ikemura
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Naoki
authors.affiliation: Department of Pulmonary Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Betsuyaku
mesh_terms: D020413:3' Untranslated Regions; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D001665:Binding Sites; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis / Q000235:genetics* / Q000401:mortality; D045744:Cell Line, Tumor; D062445:Claudin-1 / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000401:mortality; D008297:Male; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D008875:Middle Aged; D009154:Mutation; D009367:Neoplasm Staging; D011379:Prognosis; D034622:RNA Interference; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; D062445:Claudin-1; C540933:MIRN375 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D066246:ErbB Receptors
keywords: Claudin-1; Migration; Non-small-cell lung cancer; Survival; Target; miR-375