Locked nucleic acid in situ hybridization analysis of miR-21 expression during colorectal cancer development.
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.
Yamamichi
Nobutake
N
Department of Microbiology and Immunology, Division of Host-Parasite Interaction, Faculty of Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Shimomura
Ryoichi
R
Inada
Ken-ichi
K
Sakurai
Kouhei
K
Haraguchi
Takeshi
T
Ozaki
Yuka
Y
Fujita
Shuji
S
Mizutani
Taketoshi
T
Furukawa
Chihiro
C
Fujishiro
Mitsuhiro
M
Ichinose
Masao
M
Shiogama
Kazuya
K
Tsutsumi
Yutaka
Y
Omata
Masao
M
Iba
Hideo
H
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*
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