title: Locked nucleic acid in situ hybridization analysis of miR-21 expression during colorectal cancer development.
abstract: OBJECTIVE
  To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using in situ hybridization (ISH).
  
  
  METHODS
  Locked nucleic acid (LNA)/DNA probes and a biotin-free tyramide signal amplification system were used in ISH analyses of miRNA expression. Conditions for specific detection of miR-21 were determined using human cell lines and miR-21-expressing lentiviral vectors. Expression was determined in 39 surgically excised colorectal tumors and 34 endoscopically resected colorectal polyps.
  
  
  RESULTS
  In the surgical samples, miR-21 expression was much higher in colorectal cancers than in normal mucosa. Strong miR-21 expression was also observed in cancer-associated stromal fibroblasts, suggesting miR-21 induction by cancer-secreted cytokines. Protein expression of PDCD4, a miR-21 target, was inversely correlated with miR-21 expression, confirming that miR-21 is indeed a negative regulator of PDCD4 in vivo. In the endoscopic samples, miR-21 expression was very high in malignant adenocarcinomas but was not elevated in nontumorigenic polyps. Precancerous adenomas also frequently showed miR-21 up-regulation.
  
  
  CONCLUSIONS
  Using the LNA-ISH system for miRNA detection, miR-21 was detectable in precancerous adenomas. The frequency and extent of miR-21 expression increased during the transition from precancerous colorectal adenoma to advanced carcinoma. Expression patterns of miR-21 RNA and its target, tumor suppressor protein PDCD4, were mutually exclusive. This pattern may have clinical application as a biomarker for colorectal cancer development and might be emphasized by self-reinforcing regulatory systems integrated with the miR-21 gene, which has been previously shown in cell culture.
authors.affiliation: Department of Microbiology and Immunology, Division of Host-Parasite Interaction, Faculty of Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
authors.forename: Nobutake
authors.initials: N
authors.lastname: Yamamichi
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Shimomura
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Inada
authors.forename: Kouhei
authors.initials: K
authors.lastname: Sakurai
authors.forename: Takeshi
authors.initials: T
authors.lastname: Haraguchi
authors.forename: Yuka
authors.initials: Y
authors.lastname: Ozaki
authors.forename: Shuji
authors.initials: S
authors.lastname: Fujita
authors.forename: Taketoshi
authors.initials: T
authors.lastname: Mizutani
authors.forename: Chihiro
authors.initials: C
authors.lastname: Furukawa
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Fujishiro
authors.forename: Masao
authors.initials: M
authors.lastname: Ichinose
authors.forename: Kazuya
authors.initials: K
authors.lastname: Shiogama
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Tsutsumi
authors.forename: Masao
authors.initials: M
authors.lastname: Omata
authors.forename: Hideo
authors.initials: H
authors.lastname: Iba
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D015342:DNA Probes / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization; D035683:MicroRNAs / Q000032:analysis / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics; D011230:Precancerous Conditions / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D015342:DNA Probes; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D009841:Oligonucleotides; C114487:PDCD4 protein, human; D016601:RNA-Binding Proteins; C477371:locked nucleic acid